Cosmid Vector for Transforming Plant and Use Thereof

ABSTRACT

The present invention provides novel cosmid vectors for plant transformation. The cosmid vectors have a full length of 15 kb or less and contain: 1) an origin of replication of an IncP plasmid, but not any origin of replication of other plasmid groups; 2) the trfA1 gene of an IncP plasmid; 3) an oriT of an IncP plasmid; 4) the incC1 gene of an IncP plasmid; 5) a cos site of lambda phage, which is located outside the T-DNA; 6) a drug resistance gene expressed in  E. coli  and a bacterium of  Agrobacterium ; 7) a T-DNA right border sequence of a bacterium of  Agrobacterium ; 8) a T-DNA left border sequence of a bacterium of  Agrobacterium ; 9) a selectable marker gene for plant transformation located between 7) and 8) and expressed in a plant; and 10) restriction endonuclease recognition site(s) located between 7) and 8) for cloning a foreign gene.

This application is a Divisional of co-pending application Ser. No.12/306,163 filed on Mar. 16, 2009 and for which priority is claimedunder 35 U.S.C. §120. application Ser. No. 12/306,163 is the nationalphase of PCT International Application No. PCT/JP2007/062720 filed onJun. 25, 2007 under 35U.S.C. §371. PCT International Application No.PCT/JP2007/062720 claims the benefit of priority of PCT/JP2006/312633filed on Jun. 23, 2006. The entire contents of each of theabove-identified applications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to novel cosmid vectors for transformingplant and use thereof.

BACKGROUND ART

Various vectors have been previously developed for the purpose of planttransformation.

Recently, the entire genome sequences of Arabidopsis thaliana and rice(Oryza sativa) were elucidated, which moved the focus of plant genomestudies from the accumulation of nucleotide sequence information to theelucidation of gene functions. For the elucidation of gene functions,experiments are absolutely necessary in which cloned DNA is transferredinto a plant to analyze changes in the phenotype. If large DNA could betransferred in this operation, the study efficiency would bedramatically improved.

Thus, a number of vectors intended to transfer large DNA fragments intoplants were developed. As typical examples, cosmid vectors for planttransformation were prepared, such as pOCA18 (Olszewski et al., 1988,Nucleic Acids Res. 16: 10765-10782) and pLZO3 (Lazo et al., 1991,Bio/Technology 9: 963-967). The use of a cosmid has the advantage that alambda phage packaging reaction can be used, which allows easy cloningof relatively large genomic fragments (Sambrook J. and Russell D. W.2001. Molecular Cloning, A Laboratory Manual, 3rd edn. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA.). In cloningwith a cosmid vector and a packaging reaction, the total size of thevector and the insert fragment is 40 kb-50 kb so that the size of theinsert fragment is restricted within a certain range by the size of thevector and the sizes of the vector and the insert fragment inverselycorrelate with each other.

Vectors such as pOCA18 and pLZO3 contain elements for planttransformation such as T-DNA border sequences and a selectable marker(kanamycin resistance gene) in pRK290 (Ditta et al., 1980, Proc. Natl.Acad. Sci. USA 77: 7347-7351) which is a typical vector having an originof replication (oriV) of an IncP plasmid that is functional in both E.coli and Agrobacterium. These vectors per se had a size of 24.3-30.1 kb,and therefore, the size of DNA that can be cloned using a packagingreaction was about 20 kb (pOCA18), or about 13-22 kb (pLZO3) on average.These vectors have an origin of replication (oriV) of an IncP plasmid,but other vectors such as pCIT103 and pCIT104 (Ma et al. 1992 Gene 117:161-167) have an origin of replication from ColE1 in addition to anorigin of replication (oriV) of an IncP plasmid. On the other hand, pC22(Simoens et al. 1986 Nucleic Acids Res 14: 8073-8090) is a vector havingan origin of replication from ColE1 and an origin of replication from anRi plasmid. Other cosmid vectors capable of plant transformation includepMON565 (Klee et al. 1987 Mol Gen Genet 210: 282-287) and pCLD04541(Bent et al. 1994 Science 265: 1856-1860), but they are not suitable forcloning DNA fragments of 25 kb or more because their own sizes are 24 kband 29 kb, respectively. Other examples such as pE4cos(16 kb, Klee etal. 1987 Mol Gen Genet 210: 282-287), pMON565, pLZ03, pOCA18, pCLD04541,pC22 and the like had a structure containing a cos site within theT-DNA.

Subsequently, the BIBAC vector (binary bacterial artificial chromosome,Hamilton U.S. Pat. No. 5,733,744, Hamilton et al., 1996, Proc. Natl.Acad. Sci. USA 93:9975-9979, Hamilton, 1997, Gene 200:107-116) wasdeveloped, which is capable of cloning DNA fragments of up to about 150kb and transferring them into plants. This vector is based on a BACvector capable of carrying large DNA fragments and further containselements for plant transformation such as T-DNA border sequences and aselectable marker as well as an origin of replication for Agrobacterium.The TAC vector (transformation-competent bacterial artificialchromosome) pYLTAC7 (Liu et al., 1999, Proc. Natl. Acad. Sci. USA 96:6535-6540.) was also developed, which is capable of cloning DNA of up toabout 80 kb and transferring it into plants. This vector is based on ahigh-capacity PAC vector (P1-derived artificial chromosome) using thereplication mechanism of P1 phage and contains elements for planttransformation such as T-DNA border sequences and a selectable marker aswell as an origin of replication for Agrobacterium. These vectorscontain an origin of replication of (ori) from a plasmid existing as asingle copy per cell in E. coli and Agrobacterium for the purpose ofstably maintaining a large foreign gene. That is, they use an F factoron (BIBAC) or a P1 phage on (TAC) as on for E. coli and an R1 on fromAgrobacterium rhizogenes (both BIBAC and TAC) as on for Agrobacterium.However, the use of an origin of replication from a single-copy plasmidis not necessarily essential, and vectors having an origin ofreplication (oriV) of an IncP plasmid known to exist as a few copies percell such as pSLJ1711 and pCLD04541 were reported to be capable ofstably maintaining plant genomic DNA fragments of more than 100 kb insize (Tao and Zhang (1998) Nucleic Acids Res 26: 4901-4909). Inaddition, pBIGRZ was also reported, which contains R1 on in theversatile binary plasmid vector pBI121 (JPA Hei-10-155485).

Such vectors can be used to clone large DNA fragments far exceeding 50kb, but involve complicated cloning operations. Cloning of large DNArequires skilled techniques and a considerable amount of time and labor.Transformation with BIBAC requires special Agrobacterium cellsoverexpressing virG or the like and results in a much lowertransformation efficiency (the number of selected calli/inoculated leafsection) for fragments of 150 kb as compared with those of normal smallvectors (Hamiltin et al., 1996, Proc Natl Acad Sci USA 93: 9975-9979,Shibata and Liu, 2000, Trend Plant Sci 5: 354-357). Thus, transformationof large fragments into plants with BIBAC or TAC is limited to a fewspecific examples of large fragments (e.g., Hamiltin et al., 1996, ProcNatl Acad Sci USA 93: 9975-9979, Liu et al., 1999, Proc Natl Acad SciUSA 96: 6535-6540, Lin et al., 2003, Proc Natl Acad Sci USA 100:5962-5967, Nakano et al., 2005, Mol Gen Genomics 273: 123-129).

As described above, pCLD04541 is a cosmid of 29 kb in size, andtherefore, the size of DNA fragments that can be cloned using a lambdaphage packaging reaction is 10-20 kb. If cloning of larger DNA fragmentsis intended, a packaging reaction cannot be used as described above, andthus complicated cloning operations and a considerable amount of timeand labor are required.

Recently, genetic markers based on DNA sequence polymorphisms orso-called DNA markers are used more and more frequently with the advancein genome studies of higher plants. Many attempts have been made toclone unknown genes of higher plants known only by their phenotypes onthe basis of genetic map information using DNA markers, i.e., so-calledmap-based cloning. Generally, the basic protocol of map-based cloning isas follows.

1. Examine a relatively small segregating population with a set of DNAmarkers widely used for rough mapping of a candidate region on achromosome.

2. Screen a large segregating population with a set of DNA markers newlydesigned for the particular region of the genome to narrow down thecandidate region.

3. Determine the nucleotide sequence of the genetic region and guess acandidate gene.

4. Transfer a DNA fragment containing the candidate gene into a plantand determine the effect/function of the gene on the basis of thephenotype.

Many previous successful cases often involve narrowing down the geneticregion to about 1-3 genes in step 3 and transferring several DNAfragments of several kilobases or less in step 4. However, it is notalways easy to narrow down the genetic region. For example, it is oftenimpossible to narrow down the genetic region to 150 kb or less inchromosomal regions near centromeres because of the low frequency ofgenetic recombination upon cross-hybridization. Even cases wherenarrowing down is possible often require repeating the operation of step2 and therefore enormous amounts of time. Even if narrowing down toabout 50 kb were possible, it would be very difficult to guess acandidate gene without strong information linking the phenotype to thegene sequence in step 3.

Thus, map-based cloning is relatively easy until the step of defining acandidate region including one to a few DNA fragments cloned by a BACvector by narrowing down to some extent (to 50 kb to several hundreds ofkilobases), but it is often technically difficult to further pursue theanalysis to practically identify a gene, and even if it is possible,enormous amounts of labor and time are often required.

REFERENCES

-   Patent Publication No. 1: U.S. Pat. No. 5,733,744-   Patent Publication No. 2: Japanese Patent Laid-open Publication No.    H10-155485-   Patent Publication No. 3: WO2005/040374-   Non-patent Publication No. 1: Olszewski et al., 1988, Nucleic Acids    Res. 16: 10765-10782-   Non-patent Publication No. 2: Lazo et al., 1991,-   Bio/Technology 9: 963-967-   Non-patent Publication No. 3: Ditta et al., 1980, Proc. Natl. Acad.    Sci. USA 77: 7347-7351-   Non-patent Publication No. 4: Ma et al. 1992 Gene 117: 161-167-   Non-patent Publication No. 5: Simoens et al. 1986 Nucleic Acids Res    14: 8073-8090-   Non-patent Publication No. 6: Klee et al. 1987 Mol Gen Genet 210:    282-287-   Non-patent Publication No. 7: Bent et al. 1994 Science 265:    1856-1860-   Non-patent Publication No. 8: Hamilton et al., 1996, Proc. Natl.    Acad. Sci. USA 93:9975-9979,-   Non-patent Publication No. 9: Hamilton, 1997, Gene 200:107-116-   Non-patent Publication No. 10: Liu et al., 1999, Proc. Natl. Acad.    Sci. USA 96: 6535-6540-   Non-patent Publication No. 11: Tao and Zhang, 1998, Nucleic Acids    Res 26: 4901-4909-   Non-patent Publication No. 12: Shibata and Liu, 2000, Trend Plant    Sci 5: 354-357-   Non-patent Publication No. 13: Lin et al., 2003, Proc Natl Acad Sci    USA 100: 5962-5967,-   Non-patent Publication No. 14: Nakano et al., 2005, Mol Gen Genomics    273: 123-129-   Non-patent Publication No. 15: Pansegrau et al. (1994) J Mol Biol    239: 623-663-   Non-patent Publication No. 16: Knauf and Nester 1982 Plasmid 8:    45-54-   Non-patent Publication No. 17: Komari et al. 1996 Plant J 10:165-174-   Non-patent Publication No. 18: Zambryski et al. 1980 Science 209:    1385-1391-   Non-patent Publication No. 19: Schmidhauser and Helinski, J.    Bacteriol. 164:446-455, 1985-   Non-patent Publication No. 20: Winans et al. 1986 Proc. Natl. Acad.    Sci. USA 83: 8278-8282-   Non-patent Publication No. 21: Pazour et al. 1992 J. Bac    174:4169-4174-   Non-patent Publication No. 22: Ward et al. (1988) J Biol Chem 263:    5804-5814-   Non-patent Publication No. 23: Frame et al. 2002 Plant Physiol 129:    13-22-   Non-patent Publication No. 24: Hansen et al. 1994 ProNAS    91:7603-7607-   Non-patent Publication No. 25: Ishida et al. 1996 Nat Biotechnol    14:745-50-   Non-patent Publication No. 26: Close et al. 1984 Plasmid 12: 111-118-   Non-patent Publication No. 27: Jin et al. 1987 J Bacteriol 169:    4417-4425-   Non-patent Publication No. 28: Wang et al. 2000 Gene 242: 105-114-   Non-patent Publication No. 29: Okumura and Kado (1992) Mol Gen Genet    235: 55-63-   Non-patent Publication No. 30: Christensen et al. 1992 Plant Mol    Biol 18: 675-689-   Non-patent Publication No. 31: Bilang et al. (1991) Gene 100:    247-250-   Non-patent Publication No. 32: Hirsch and Beringer 1984 Plasmid 12:    139-141-   Non-patent Publication No. 33: Konieczny and Ausubel 1993 Plant    Journal 4: 403-410-   Non-patent Publication No. 34: Hiei et al. (1994) Plant J 6: 271-282-   Non-patent Publication No. 35: Ishida et al. (2003) Plant    Biotechnology 20:57-66-   Non-patent Publication No. 36: Hiei and Komari (2006) Plant Cell,    Tissue and Organ Culture 85: 271-283-   Non-patent Publication No. 37: Komori et al. (2004) Plant J 37:    315-325-   Non-patent Publication No. 38: Kazama and Toriyama (2003) FEBS lett    544: 99-102.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

WO2005/040374, which is incorporated by reference herein in itsentirety, discloses a method for efficiently selecting and preparing anumber of genomic DNA fragments capable of improving traits expressed inheterosis or quantitative traits as cloned DNA fragments. We haveselected large genomic DNA fragments capable of introducingagriculturally useful mutations by using the method described inWO2005/040374. However, the success rate of transferring clones carriedin E. coli into Agrobacterium was about 80%. Moreover, only about 60% ofAgrobacterium strains harboring clones was able to transform plants. Inview of this result, we examined whether or not the efficiency of themethod of WO2005/040374 could be significantly improved by changing thevector used. However, the efficiency of this method could not beimproved by any vector ever known.

Thus, an object of the present invention is to provide a novel vectorcapable of improving the efficiency of selecting and cloning relativelylarge genomic DNA fragments, e.g., in the method described inWO2005/040374.

Another object of the present invention is to provide a vectorpreferably fulfilling all of the requirements below:

-   -   it allows efficient cloning of DNA fragments of about 25-40 kb        in size;    -   it is stably maintained in E. coli and Agrobacterium cells;    -   it can be efficiently introduced into Agrobacterium;    -   the copy number per cell in E. coli and Agrobacterium is 4-5;        and    -   it allows efficient transfer of only cloned DNA fragments of        interest into plants, preferably monocotyledons.

Still another object of the present invention is to provide a genetransfer method for transferring a gene into a plant at a very highefficiency using such a vector.

Still another object of the present invention is to provide a method forrapidly narrowing down a gene region for completing map-based cloningwith ease in a short time using such a vector.

Still another object of the present invention is to provide a plasmidcapable of further improving the transformation efficiency by combiningit with said vector.

Means for Solving the Problems

Cosmid Vectors

The cosmid vectors of the present invention are vectors having a fulllength of 15 kb or less satisfying all of the following criteria(hereinafter referred to as “pLC vectors”):

1) they contain an origin of replication (oriV) of an IncP plasmid, butdo not contain any origin of replication of other plasmid groups;

2) they contain the trfA1 gene of an IncP plasmid;

3) they contain an origin of conjugative transfer (oriT) of an IncPplasmid;

4) they contain the incC1 gene of an IncP plasmid;

5) they contain a cos site of lambda phage and the cos site is locatedoutside the T-DNA;

6) they contain a drug resistance gene expressed in E. coli and abacterium of the genus Agrobacterium;

7) they contain a T-DNA right border sequence of a bacterium of thegenus Agrobacterium;

8) they contain a T-DNA left border sequence of a bacterium of the genusAgrobacterium;

9) they contain a selectable marker gene for plant transformationlocated between 7) and 8) and expressed in a plant; and

10) they contain restriction endonuclease recognition site(s) locatedbetween 7) and 8) for cloning a foreign gene.

The vectors of the present invention are cosmid vector containing a cossite of lambda phage. This allows cloning of relatively large genomicfragments by a packaging reaction (Sambrook J. and Russell D. W. 2001.Molecular Cloning, A Laboratory Manual, 3rd edn. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA.). In cloning using apackaging reaction of a cosmid vector, the total size of the vector andthe insert fragment is around 40 kb-50 kb so that the size of the insertfragment is restricted within a certain range by the size of the vector.The vectors of the present invention have a full length of 15 kb orless, preferably 12-14 kb because they are intended to clone a DNAfragment of up to about 25-40 kb, preferably 30-40 kb.

1) An origin of replication (oriV) of an IncP plasmid: The oriV isfunctional in both E. coli and Agrobacterium. The nucleotide sequence ofthe oriV of the present invention is not specifically limited so far asit has the function of oriV, i.e., the function of an origin ofreplication of an IncP plasmid.

The oriV has molecular biological properties described in detail inPansegrau et al. (1994) J Mol Biol 239: 623-663, and it is defined asnucleotides 12200-12750 of the sequence of Genbank/EMBL Accession NumberL27758 (full length 60099 bp). This corresponds to nucleotides 3451-4002of SEQ ID NO: 1 (core sequence of oriV).

The oriV can be conventionally prepared from an IncP plasmid such aspVK102 (Knauf and Nester 1982 Plasmid 8: 45-54). For example, a 0.9 kbDNA (nucleotides 3345-4247 of SEQ ID NO: 1) amplified by PCR from pVK102can be used as the oriV.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 3451-4002, more preferably 3345-4247 of SEQ ID NO: 1described above under stringent conditions and having the function oforiV can also be used. Alternatively, a nucleic acid containing anucleotide sequence having an identity of at least 95%, more preferably97%, still more preferably 99% to the nucleotide sequence of nucleotides3451-4002, more preferably 3345-4247 of SEQ ID NO: 1 described above andhaving the function of oriV can also be used.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 3451-4002 of SEQ ID NO: 1 may be selected as a sequencehaving a similar function. We investigated from various viewpoints thereason why the final transformation efficiency was only about 50% (80% x60%=48%) when the method described in WO2005/040374 was used, andconcluded that this might be ascribable to the use of the cloning vectorpSB200.

A replication origin of pSB200 is from ColE1, and plasmids having anorigin of replication from ColE1 exist in a relatively high copy number,i.e., 30-40 copies per E. coli cell. Tao and Zhang (1998, Nucleic AcidsRes 26: 4901-4909) assume that E. coli can stably maintain 1200-1500 kbof foreign DNA per cell. If 30-40 kb of DNA is cloned by pSB200, thetotal DNA amount including the vector size reaches 1200-2000 kb percell, which may exceed the range assumed above. Another possible reasonis that pSB200 is a plasmid that is not replicated alone inAgrobacterium. Thus, a vector having an origin of replication (oriV) ofan IncP plasmid called pSB1 is preliminarily introduced intoAgrobacterium, and a cointegrate between pSB200 and pSB1 is prepared viahomologous recombination between DNA sequences contained in both pSB200and pSB1, thereby introducing pSB200 into Agrobacterium. It isundeniable that some adverse phenomenon could occur during such anoperation to result in the failure in the transfer of pSB200.

If the copy number in E. coli and Agrobacterium is too low, however, theanalysis of DNA or the like will be inefficient.

Based on the foregoing discussion, we prepared and tested vectorscontaining an origin of replication (oriV) of an IncP plasmid that isfunctional in both E. coli and Agrobacterium but not any origin ofreplication of other plasmid groups and existing in 4-5 copies in thesebacteria. As a result, we found that the transformation efficiency isimproved by using such vectors in plant transformation, specificallye.g., in the method described in WO2005/040374, and thus achieved thepresent invention.

2) The trfA1 gene of an IncP plasmid: The trfA1 gene is important as atransacting replication factor of IncP plasmids and necessary for anoriV to perform its function. The nucleotide sequence of the trfA1 geneof the present invention is not specifically limited so far as it hasthe function of trfA1, i.e. the function of a transacting replicationfactor.

It has molecular biological properties described in detail in Pansegrauet al. (1994) J Mol Biol 239: 623-663, and it is defined as nucleotides16521-17669 of the sequence of Genbank/EMBL Accession Number L27758(full length 60099 bp). This corresponds to nucleotides 6323-7471 of SEQID NO: 1 (core sequence of trfA1).

TrfA1 can be conventionally prepared from an IncP plasmid such as pVK102(Knauf and Nester 1982 Plasmid 8: 45-54). For example, a 3.2 kb DNAfragment (nucleotides 5341-8507 of SEQ ID NO: 1) amplified by PCR frompVK102 can be used as the trfA1 gene.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 6323-7471, more preferably 5341-8507 of SEQ ID NO: 1described above under stringent conditions and having the function ofthe trfA1 gene can also be used. Alternatively, a nucleic acidcontaining a nucleotide sequence having an identity of at least 95%,more preferably 97%, still more preferably 99% to the nucleotidesequence of nucleotides 6323-7471, more preferably 5341-8507 of SEQ IDNO: 1 described above and having the function of the trfA1 gene can alsobe used.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 6323-7471 of SEQ ID NO: 1 may be selected as a sequencehaving a similar function.

3) An origin of conjugative transfer (oriT) of an IncP plasmid: oriT isan element responsible for conjugation (mating). One of the purposes ofthe vectors of the present invention is to perform large-scale andhigh-efficient transformation. For that purpose, conjugation (mating)between E. coli and Agrobacterium is necessary, and oriT contributes tothe conjugation (mating). The sequence of the oriT of the presentinvention is not specifically limited so far as it has the function oforiT, i.e. the function of an element responsible for conjugation(mating).

The oriT has molecular biological properties described in detail inPansegrau et al. (1994) J Mol Biol 239: 623-663, and it is defined asnucleotides 51097-51463 of the sequence of Genbank/EMBL Accession NumberL27758 (full length 60099 bp). The oriT can be conventionally preparedfrom an IncP plasmid such as pVK102 (Knauf and Nester 1982 Plasmid 8:45-54). For example, a 0.8 kb DNA fragment (nucleotides 1-816 of SEQ IDNO: 1) amplified by PCR from pVK102 can be used as the oriT.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 1-816 of SEQ ID NO: 1 described above under stringentconditions and having the function of oriT can also be used.Alternatively, a nucleic acid containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 1-816 of SEQ ID NO: 1described above and having the function of oriT can also be used.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 1-816 of SEQ ID NO: 1 may be selected as a sequencehaving a similar function.

4) The incC1 gene of an IncP plasmid: The incC1 gene contributes to thestability of IncP plasmids. The nucleotide sequence of the incC1 gene ofthe present invention is not specifically limited so far as it has thefunction of the incC1 gene contributing the stability of IncP plasmids.

This gene has molecular biological properties described in detail inPansegrau et al. (1994) J Mol Biol 239: 623-663, and it is defined asnucleotides 58260-59354 of the sequence of Genbank/EMBL Accession NumberL27758 (full length 60099 bp). This corresponds to nucleotides 1179-2273of SEQ ID NO: 1 (core sequence of the incC1 gene).

IncC1 can be conventionally prepared from an IncP plasmid such as pVK102(Knauf and Nester 1982 Plasmid 8: 45-54). For example, a 2.1 kb DNAfragment (nucleotides 817-2935 of SEQ ID NO: 1) amplified by PCR frompVK102 can be used as the incC1 gene.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 1179-2273, more preferably 817-2935 of SEQ ID NO: 1described above under stringent conditions and having the function ofthe incC1 gene can also be used. Alternatively, a nucleic acidcontaining a nucleotide sequence having an identity of at least 95%,more preferably 97%, still more preferably 99% to the nucleotidesequence of nucleotides 1179-2273, more preferably 817-2935 of SEQ IDNO: 1 described above and having the function of the incC1 gene can alsobe used.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 1179-2273 of SEQ ID NO: 1 may be selected as a sequencehaving a similar function.

5) A cos site of lambda phage: The vectors of the present inventioncontain a cos site of lambda phage to utilize the packaging reaction ofcosmid vectors. The nucleotide sequence of the cos site of lambda phageof the present invention is not specifically limited so far as it hasthe function of a cos site of lambda phage, i.e. the functioncontributing to the packaging reaction of cosmid vectors.

The cos site of lambda phage has molecular biological propertiesdescribed in detail in Sambrook J. and Russell D. W. (2001), and it hasthe sequence 5′-aggtcgccgccc-3′ (SEQ ID NO: 9) (the core sequence of acos site of lambda phage). The cos can be conventionally prepared from aplasmid such as pSB11 (Komari et al. 1996 Plant J 10:165-174). Forexample, a 0.4 kb DNA fragment (nucleotides 2936-3344 of SEQ ID NO: 1)amplified by PCR from pSB11 can be used.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence of SEQID NO: 9 described above, more preferably the nucleotide sequence ofnucleotides 2936-3344 of SEQ ID NO: 1 under stringent conditions andhaving the function of a cos site of lambda phage can also be used.Alternatively, a nucleic acid containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of SEQ ID NO: 9 described above, morepreferably the nucleotide sequence of nucleotides 2936-3344 of SEQ IDNO: 1 and having the function of a cos site of lambda phage can also beused.

The cos site should be located outside the T-DNA because undesired DNAwill be introduced into plants if the cos site is located inside theT-DNA.

6) The drug resistance gene expressed in E. coli and a bacterium of thegenus Agrobacterium is used as a selectable marker for transformation.This drug resistance gene confers e.g., antibiotic resistance orautotrophy, including, but not limited to, a kanamycin resistance gene,a spectinomycin resistance gene, an ampicillin resistance gene, atetracycline resistance gene, a gentamycin resistance gene, a hygromycinresistance gene, etc.

7), 8) T-DNA right border sequence (RB) and left border sequence (LB) ofa bacterium of the genus Agrobacterium are essential for transformation(Zambryski et al. 1980 Science 209: 1385-1391), and a cloning site for aforeign gene is located between them. The nucleotide sequences of the RBand LB of the present invention are not specifically limited so far asthey have the function of T-DNA right border sequence (RB) and leftborder sequence (LB) of a bacterium of the genus Agrobacterium. They canbe each conventionally prepared from a plasmid such as pSB11 (Komari etal. 1996 Plant J 10:165-174). For example, nucleotides 13253-13277 and3479-3503 of SEQ ID NO: 2 can be used, respectively.

Alternatively, nucleic acids containing nucleotide sequences hybridizingto complementary strands of the nucleotide sequences of nucleotides13253-13277 and 3479-3503 of SEQ ID NO: 2 described above understringent conditions and having the functions of the RB and LB,respectively, can also be used. Alternatively, nucleic acids containingnucleotide sequences having an identity of at least 95%, more preferably97%, still more preferably 99% to the nucleotide sequences ofnucleotides 13253-13277 and 3479-3503 of SEQ ID NO: 2 described aboveand having the functions of the RB and LB, respectively, can also beused.

It will be recognized by those skilled in the art that shorter regionsin nucleotides 13253-13277 and 3479-3503 of SEQ ID NO: 2 may be selectedas sequences having similar functions.

9) A selectable marker gene for plant transformation expressed in aplant cell and located between 7) and 8) is included. The selectablemarker gene for plant transformation is not specifically limited, andknown selectable marker genes can be used. Preferably, it is any one ofa hygromycin resistance gene, a phosphinotricin resistance gene, and akanamycin resistance gene. For use in transformation of monocotyledons,a hygromycin resistance gene or a phosphinotricin resistance gene ispreferred.

10) Restriction endonuclease recognition site(s) located between 7) and8) for cloning a foreign gene are included. The restriction endonucleaserecognition sites for cloning a foreign gene are not specificallylimited, and known restriction endonuclease recognition sites can beused, but the same recognition sites are desirably absent elsewhere onthe vectors.

In the cosmid vector constructs of the present invention, the order ofall of the seven elements consisting of elements 1)-6) and a unit of7)-10) is not limited. Moreover, the order of 9) and 10) located between7) and 8) is not limited.

The cosmid vectors of the present invention preferably satisfy one ormore of the following criteria A-G.

A. The nucleotide sequence of oriV in 1) comprises the followingnucleotide sequence:

i) the nucleotide sequence of nucleotides 3451-4002, more preferably3345-4247 of SEQ ID NO: 1;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides3451-4002, more preferably 3345-4247 of SEQ ID NO: 1 under stringentconditions and having the function of oriV; or

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 3451-4002, more preferably3345-4247 of SEQ ID NO: 1 and having the function of oriV.

B. The trfA1 gene in 2) comprises the following nucleotide sequence:

i) the nucleotide sequence of nucleotides 6323-7471, more preferably5341-8507 of SEQ ID NO: 1;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides6323-7471, more preferably 5341-8507 of SEQ ID NO: 1 under stringentconditions and having the function of the trfA1 gene;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 6323-7471, more preferably5341-8507 of SEQ ID NO: 1 and having the function of the trfA1 gene.

C. The oriT in 3) comprises the following nucleotide sequence:

i) the nucleotide sequence of nucleotides 1-816 of SEQ ID NO: 1;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides1-816 of SEQ ID NO: 1 under stringent conditions and having the functionof oriT;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 1-816 of SEQ ID NO: 1 andhaving the function of oriT.

D. The incC1 gene in 4) comprises the following nucleotide sequence:

i) the nucleotide sequence of nucleotides 1179-2273, more preferably817-2935 of SEQ ID NO: 1;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides1179-2273, more preferably 817-2935 of SEQ ID NO: 1 under stringentconditions and having the function of the incC1 gene;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 1179-2273, more preferably817-2935 of SEQ ID NO: 1 and having the function of the incC1 gene.

E. The cos site of lambda phage in 5) comprises the following nucleotidesequence:

i) the nucleotide sequence of SEQ ID NO: 9, more preferably thenucleotide sequence of nucleotides 2936-3344 of SEQ ID NO: 1;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of SEQ ID NO: 9,more preferably the nucleotide sequence of nucleotides 2936-3344 of SEQID NO: 1 under stringent conditions and having the function of a cossite of lambda phage;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of SEQ ID NO: 9, more preferably thenucleotide sequence of nucleotides 2936-3344 of SEQ ID NO: 1 and havingthe function of a cos site of lambda phage.

F. The T-DNA right border sequence (RB) of a bacterium of the genusAgrobacterium in 7) comprises the following nucleotide sequence:

i) the nucleotide sequence of nucleotides 13253-13277 of SEQ ID NO: 2;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides13253-13277 of SEQ ID NO: 2 under stringent conditions and having thefunction of RB;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 13253-13277 of SEQ ID NO: 2and having the function of RB.

G. The T-DNA left border sequence (LB) of a bacterium of the genusAgrobacterium in 8) comprises the following nucleotide sequence:

i) the nucleotide sequence of nucleotides 3479-3503 of SEQ ID NO: 2;

ii) a nucleotide sequence containing a nucleotide sequence hybridizingto a complementary strand of the nucleotide sequence of nucleotides3479-3503 of SEQ ID NO: 2 under stringent conditions and having thefunction of LB;

iii) a nucleotide sequence containing a nucleotide sequence having anidentity of at least 95%, more preferably 97%, still more preferably 99%to the nucleotide sequence of nucleotides 3479-3503 of SEQ ID NO: 2 andhaving the function of LB.

By satisfying all of the criteria 1)-10) above for the cosmid vectors ofthe present invention, a vector fulfilling all of the requirements belowcan be prepared:

-   -   it allows efficient cloning of DNA fragments of about 25-40 kb        in size, preferably 30-40 kb;    -   it is stably maintained in E. coli and Agrobacterium cells;    -   it can be efficiently introduced into Agrobacterium;    -   the copy number per cell in E. coli and Agrobacterium is 4-5;        and    -   it allows efficient transfer of only cloned DNA fragments of        interest into plants, preferably monocotyledons.

However, the development of such a vector was not straightforward evenafter the requirements above had been defined. This is partially due tothe very complex control mechanism of plasmid replication. Specifically,the most suitable vector backbone for the requirements above is a smallplasmid (in the order of 12 kb to 15 kb) having an origin of replication(oriV) of an IncP plasmid. However, the backbone 60 kb IncP plasmid hasmany genes involved in the replication of the plasmid and partitioningduring cell division, resulting in a very complex mechanism, though itsentire nucleotide sequence has been determined (Pansegrau et al. J. Mol.Biol. 239:623-663, 1994). Thus, it is not easy to prepare a small vectorhaving an origin of replication (oriV) of an IncP plasmid and stablymaintained in bacteria. In fact, plasmids of various sizes derived fromIncP plasmids have been studied, but small plasmids are generallyunstable and widely differ in stability depending on the bacterialspecies (Schmidhauser and Helinski, J. Bacteriol. 164:446-455, 1985).pE4cos is an example of the plasmid which has lost stability inAgrobacterium by size reduction. The reasons for this have beendiscussed to a certain extent (Klee et al. 1987 Mol Gen Genet 210:282-287), but it can be hardly said that they have been clarified.

Schmidhauser and Helinski (J. Bacteriol. 164:446-455, 1985) say that“there is no universal set of genetic determinants in plasmid RK2 thataccounts for stable maintenance in all gram-negative bacteria”,indicating great difficulty in the preparation of a small and stablevector. The plasmid RK2 here (also often designated as pRK2) is one oftypical IncP plasmids. The procedure for constructing such a vectoroften uses the step of cloning elements of a backbone plasmid usinganother vector. However, DNA fragments involved in the replication ofbacterial plasmids or chromosomes are sometimes difficult to clone. Ifsuch a problem occurs, a means to solve it must be developed, whichcontributes to the difficulty in the construction of novel vectors.

Non-limitative examples of the cosmid vectors (pLC series) of thepresent invention are as follows.

i) pLC40 (SEQ ID NO: 2, FIG. 6)

A binary cosmid vector having a full length of 13429 by characterized inthat:

1) it contains an origin of replication (oriV) of an IncP plasmid, butdoes not contain any origin of replication of other plasmid groups;

2) it contains the trfA1 gene, 3) oriT, and 4) the incC1 gene of an IncPplasmid;

5) it contains a cos site of lambda phage and the cos site is locatedoutside the T-DNA;

6) it contains the drug resistance gene nptIII (kanamycin resistancegene) expressed in E. coli and a bacterium of the genus Agrobacterium;

7) it contains a T-DNA right border sequence of a bacterium of the genusAgrobacterium;

8) it contains a T-DNA left border sequence of a bacterium of the genusAgrobacterium;

9) it contains the selectable marker gene for plant transformation hpt(hygromycin resistance gene) located between 7) and 8) and expressed ina plant; and

10) it contains restriction endonuclease recognition site(s) locatedbetween 7) and 8) for cloning a foreign gene, e.g., an NspV site.

pLC40 was prepared by inserting a region containing the T-DNA region ofpSB200PcHm (FIG. 1) into p6FRG. It should be noted that p6FRG is acosmid vector of 8507 by in full length having the structure shown inFIG. 5 (SEQ ID NO: 1 in the Sequence Listing) characterized in that:

1) it contains an origin of replication (oriV) of an IncP plasmid, butdoes not contain any origin of replication of other plasmid groups;

2) it contains the trfA1 gene, oriT and the incC1 gene of an IncPplasmid, and a cos site of lambda phage;

3) it contains the drug resistance gene nptIII (kanamycin resistancegene) expressed in E. coli and a bacterium of the genus Agrobacterium.

ii) pLC40GWH (SEQ ID NO: 3, FIG. 7)

A binary cosmid vector of 13174 by in full length. It differs from pLC40by an insertion of attB1, 2 sequences and a deletion of a 317 bySspI-BalI region upstream of the RB. It was prepared by inserting aregion containing the T-DNA region of pSB200PcHmGWH (FIG. 3) into p6FRG.

iii) pLC40 bar (SEQ ID NO: 4, FIG. 8)

A binary cosmid vector of 12884 by in full length. Principal differencesof pLC40 bar from pLC40 are in that the selectable marker gene for planttransformation is bar (phosphinotricin resistance gene), and that theorientation of the selectable marker unit (ubiquitin promoter-ubiquitinintron-selectable marker gene for plant transformation) on the T-DNA isopposite. It was prepared by inserting a region containing the T-DNAregion of pSB25UNpHm (FIG. 2) into p6FRG.

iv) pLC40GWB (SEQ ID NO: 5, FIG. 9)

A binary cosmid vector of 13026 by in full length. It differs from pLC40in that the selectable marker gene for plant transformation is bar(phosphinotricin resistance gene) and that attB1, 2 sequences have beeninserted. It was prepared by inserting a region containing the T-DNAregion of pSB200PcHmGWB (FIG. 4) into p6FRG.

v) pLC40GWHkorB (SEQ ID NO: 65, FIG. 10)

A binary cosmid vector of 14120 by in full length. It differs frompLC40GWH in that it contains the nucleotide sequence of the korB gene.The korB gene is located near IncC1 described above, and contributes tothe stability of IncP plasmids as IncC1 does. The nucleotide sequence ofthe korB gene of the present invention is not specifically limited sofar as it has the function of the korB gene contributing to thestability of IncP plasmids.

This sequence has molecular biological properties described in detail inPansegrau et al. (1994) J Mol Biol 239: 623-663, and it is defined asnucleotides 57187-58263 of the sequence of Genbank/EMBL Accession NumberL27758 (full length 60099 bp). This corresponds to nucleotides 6306-7382of SEQ ID NO: 65.

The korB can be conventionally prepared from an IncP plasmid such aspVK102 (Knauf and Nester 1982 Plasmid 8: 45-54). For example, a sequenceamplified by PCR from pVK102 (nucleotides 6306-7382 of SEQ ID NO: 65)can be used as the korB gene.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 6306-7382 of SEQ ID NO: 65 under stringent conditions andhaving the function of the korB gene can be used. Alternatively, anucleic acid containing a nucleotide sequence having an identity of atleast 95%, more preferably 97%, still more preferably 99% to thenucleotide sequence of nucleotides 6306-7382 of SEQ ID NO: 65 and havingthe function of the korB gene can also be used.

vi) pLCleo (SEQ ID NO: 66, FIG. 11)

A binary cosmid vector of 14195 by in full length. It differs frompLC40GWHkorB in that it contains a PspOMI site in the multicloning site,a PI-SceI upstream of it, and an attB3 site upstream of the ubiquitinpromoter.

vii) pLC40GWHvG1 (SEQ ID NO: 7, FIG. 13)

A binary cosmid vector of 14222 by in full length. It differs frompLC40GWH in that the virG gene has been inserted. It was prepared byinserting the virG gene outside the T-DNA of pPLC40GWH.

Those skilled in the art can readily derive equivalents to the sevencosmid vectors described above, i.e.,

i) the cosmid vector pLC40 consisting of the nucleotide sequence of SEQID NO: 2;

ii) the cosmid vector pLC40GWH consisting of the nucleotide sequence ofSEQ ID NO: 3;

iii) the cosmid vector pLC40 bar consisting of the nucleotide sequenceof SEQ ID NO: 4;

iv) the cosmid vector pLC40GWB consisting of the nucleotide sequence ofSEQ ID NO: 5;

v) the cosmid vector pLC40GWHKorB consisting of the nucleotide sequenceof SEQ ID NO: 65;

vi) the cosmid vector pLCleo consisting of the nucleotide sequence ofSEQ ID NO: 66; and

vii) the cosmid vector pLC40GWHvG1 consisting of the nucleotide sequenceof SEQ ID NO: 7;

said equivalents having similar functions to those of these vectors evenif the nucleotide sequences are not completely identical. Thus, these“equivalents” are also included as preferred embodiments of the cosmidvectors of the present invention.

For example, it is thought that even if the nucleotide sequences of thecosmid vectors of the present invention i)-vii) above are modifiedespecially in parts other than the elements related to criteria 1)-10)above (e.g., oriV in criterion 1), or the trfA1 gene in criterion 2)),they perform similar functions to those of the original vectors ascosmid vectors. Moreover, more than one genes or restrictionendonuclease sites having similar functions to those of the drugresistance gene in 6), the selectable marker gene for planttransformation in 9), and the restriction endonuclease recognitionsite(s) in 10) among criteria 1)-10) are known even if the nucleotidesequences are not completely identical to the nucleotide sequences inthe cosmid vectors i)-vii), and those skilled in the art can modifythese parts as appropriate.

Therefore, an “equivalent” to each of the cosmid vectors of the presentinvention i)-vii) preferably refers to a nucleotide sequence identicalto or having an identity of at least 95% or more, 97% or more, 98% ormore or 99% or more, more preferably 99.5% or more to the nucleotidesequence of each cosmid vector in the nucleotide sequences of theelements related to criteria 1)-5) and 7)-8) of the cosmid vectors ofthe present invention, especially the core sequences in these criteriaor refers to a nucleotide sequence hybridizing to a complementary strandof the nucleotide sequence of each cosmid vector under stringentconditions, said equivalent containing a mutation elsewhere in thenucleotide sequence while having similar function and effect to those ofeach vector. More preferably, it refers to a nucleotide sequenceidentical to the nucleotide sequence of each cosmid vector in thenucleotide sequences of the elements related to criteria 1)-10) of thecosmid vectors of the present invention, especially the core sequencesin these criteria and containing a mutation elsewhere in the nucleotidesequence while having similar function and effect to those of eachvector.

The degree of mutation is not specifically limited, but the “equivalent”preferably consists of a nucleotide sequence hybridizing to acomplementary strand of the nucleotide sequence of each of cosmidvectors i)-vii) under stringent conditions. The number of nucleotidesthat can be mutated is more preferably one or more, still morepreferably one to a few (e.g., to the extent at which a mutation can beintroduced by known site-directed mutagenesis).

The “equivalent” also preferably consists of a nucleotide sequencehaving an identity of 95% or more, 97% or more, 98% or more or 99% ormore, more preferably 99.5% or more to a nucleotide sequence selectedfrom the nucleotide sequences of cosmid vectors i)-vii).

The percent identity of two nucleic acid sequences can be determined byvisual inspection and mathematical calculation, or more preferably, thecomparison is done by comparing sequence information using a computerprogram. An exemplary, preferred computer program is the GeneticsComputer Group (GCG; Madison, Wis.) Wisconsin package version 10.0program, “GAP” (Devereux et al., 1984, Nucl. Acids Res. 12: 387). This“GAP” program can be used to compare not only two nucleic acid sequencesbut also two amino acid sequences or a nucleic acid sequence and anamino acid sequence. The preferred default parameters for the “GAP”program include (1) The GCG implementation of a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted amino acid comparison matrix of Gribskovand Burgess, Nucl. Acids Res. 14: 6745, 1986 as described by Schwartzand Dayhoff, eds., “Atlas of Polypeptide Sequence and Structure”,National Biomedical Research Foundation, pp. 353-358, 1979; or othercomparable comparison matrices; (2) a penalty of 30 for each gap and anadditional penalty of 1 for each symbol in each gap for amino acidsequences, or penalty of 50 for each gap and an additional penalty of 3for each symbol in each gap for nucleotide sequences; (3) no penalty forend gaps; and (4) no maximum penalty for long gaps. Other programs usedby those skilled in the art of sequence comparison can also be used,such as, for example, the BLASTN program version 2.2.7, available foruse via the National Library of Medicine website:http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.ht ml, or the UW-BLAST 2.0algorithm. Standard default parameter settings for UW-BLAST 2.0 aredescribed at the following Internet site: http://blast.wustl.edu. Inaddition, the BLAST algorithm uses the BLOSUM62 amino acid scoringmatrix, and optional parameters that can be used are as follows: (A)inclusion of a filter to mask segments of the query sequence that havelow compositional complexity (as determined by the SEG program ofWootton and Federhen (Computers and Chemistry, 1993); also see Woottonand Federhen, 1996, Analysis of compositionally biased regions insequence databases, Methods Enzymol. 266: 554-71) or segments consistingof short-periodicity internal repeats (as determined by the XNU programof Clayerie and States (Computers and Chemistry, 1993)), and (B) astatistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance, according to the stochastic model ofKarlin and Altschul, 1990; if the statistical significance ascribed to amatch is greater than this E-score threshold, the match will not bereported.); preferred E-score threshold values are 0.5, or in order ofincreasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5,1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

Plant Transformation Methods

The present invention also provides a plant transformation method usinga cosmid vector of the present invention. Specifically, the planttransformation method of the present invention comprises transforming aplant with a bacterium of the genus Agrobacterium harboring a vectorcontaining a nucleic acid fragment of a plant inserted into a cosmidvector of the present invention.

The type of the nucleic acid fragment inserted into the cosmid vector isnot specifically limited, and any fragment can be used, such as agenomic DNA fragment, a cDNA fragment, etc. The nucleic acid fragment ispreferably a genomic DNA fragment, more preferably a genomic DNAfragment derived from a plant. The size of the DNA fragment inserted ispreferably 1 kb or more, more preferably 10 kb or more, still morepreferably 20 kb or more, still more preferably 25-40 kb, still morepreferably 30-40 kb.

The preparation and introduction of the nucleic acid fragment into thecosmid vector and other operations can be performed by a known method,e.g., the method described in WO2005/040374.

The source of the nucleic acid fragment are not specifically limited. Inthe case of plant genomic DNA fragments, preferred examples includeplants in which heterosis may occur by cross with recipient plants ofgenomic DNA fragments. When the recipient plant is Japonica rice, forexample, the donor is preferably a wild species of rice Oryza rufipogonor Indica rice. When the recipient plant is a specific variety of maize,preferred examples of donor plants include the other varieties of maizeand wild species of teosinte. In general, higher heterosis has beenobserved between more distantly related plants.

The recipient plant used for transformation may belong to a differentspecies from that of the donor plant of the genomic DNA or a differentvariety of the same species or the same variety of the same species.Preferred examples of plants include substantially unrestricted widerange of plants, e.g., cereals such as rice, barley, wheat, maize,sorghum, or millet such as an extremely early maturing variety ofItalian millet or pearl millet; industrial crops such as sugar cane;pasture grasses such as Sudan grass or rose grass; plants for producingluxury grocery items such as coffee, cocoa, tea and tobacco; vegetables;fruits; ornamental plants such as flowers; weeds such as Arabidopsis,etc.

The cosmid vectors of the present invention were obtained especially toimprove the efficiency of Agrobacterium-mediated transformation amongbiological transfer methods. Therefore, the plant transformation methodis preferably Agrobacterium-mediated. However, other known planttransformation methods are not excluded. For example, known methodsinclude physical transfer methods such as microinjection,electroporation, particle gun, silicon carbide-mediated method and airinjection; and chemical transfer methods such as polyethyleneglycol-mediated method.

The type of Agrobacterium strain is not specifically limited so far asit has an antibiotic resistance other than the antibiotic resistance(gene) for the bacterium used for the construction of the vector, andknown strains such as LBA4404, A281, BHA105, PC2760, etc. can be used.

Map-Based Cloning Method

The present invention also provides an efficient map-based cloningmethod using a cosmid vector of the present invention as describedabove. The map-based cloning method is characterized in that itcomprises the steps of:

1) partially or completely digesting BAC clones containing candidategenes responsible for a plant phenotype with a restriction endonuclease;

2) subcloning DNA fragments obtained in step 1) using a cosmid vector toconstruct a library; and

3) individually transferring clones constituting the library into aplant to evaluate the phenotypes of transformed plants.

In this map-based cloning method, the DNA fragments obtained in step 1)preferably have a size of, but not limited to, 25-40 kb. Morepreferably, the cosmid vector in 2) is a cosmid vector as described inthe section “Cosmid vectors” above.

The “candidate genes” refer to a group of genes including genes likelyto be responsible for a plant phenotype. The “plant phenotype” is notspecifically limited, but includes various agriculturally usefulphenotypes such as high vigor of the whole plant, large sizes of theplant and organs, high yield, high growth speed, disease and insectresistance, resistance to various environmental stresses such asdrought, high temperature, low temperature, etc., an increase ordecrease of a specific component, an increase or decrease of a specificenzyme activity, dwarfness, etc.

For example, suppose that candidate genes were found to be contained inDNA fragments carried by more than one BAC clones of 100-200 kb. Then,these cloned DNAs are partially or completely digested with anappropriate restriction endonuclease to prepare overlapping fragments ofabout 40 kb, which are then subcloned using a transformation vector ofthe present invention. It is not necessary to investigate in detail therelative positions and the overlapping of the subcloned DNA fragments.According to a statistical calculation, any site on original fragmentsin 200 kb clones is maintained by randomized 21 subclones with a 99%probability (e.g., see [0043]-[0047] in WO2005/040374).

Then, each subclone is transferred into a plant to prepare about 10independent transformants per subclone, and the effect of the gene isanalyzed. According to this operation, the candidate region can be firstnarrowed down to 40 kb by identifying subclones containing candidategenes, and then the candidate region can be further restricted to a verynarrow region by comparing the experimental results between adjacentsubclones. Thus, the efficiency of identifying candidate genes greatlyimproves.

Transformation Method Additionally Using the virG Gene (and the virBGene)

In a preferred embodiment, the plant transformation method of thepresent invention is characterized in that it uses a bacterium of thegenus Agrobacterium harboring the following elements:

1a) a vector containing a nucleic acid fragment of a plant and the virGgene of a bacterium of the genus Agrobacterium inserted into the cosmidvector of the present invention; or

1b) a vector containing a nucleic acid fragment of a plant inserted intothe cosmid vector of the present invention, and a plasmid capable ofcoexisting with an IncP plasmid in a cell of a bacterium of the genusAgrobacterium and containing the virG gene of a bacterium of the genusAgrobacterium, and

2) a Ti plasmid or Ri plasmid of a bacterium of the genus Agrobacterium.

virG is one of vir genes of Agrobacterium that play a role in thetransfer of the T-DNA into plants, and it is regarded as a transcriptionfactor of the virB gene or the like (Winans et al. 1986 Proc. Natl.Acad. Sci. USA 83: 8278-8282). As an example of virG, virGN54D is avariant in which the amino acid at position 54 of the virG protein ischanged from asparagine to aspartic acid to increase the expression ofthe virB gene as compared with the wild-type virG (Pazour et al. 1992 J.Bac 174:4169-4174). In the present transformation method, the virG geneis preferably virGN54D.

In an embodiment of the present invention 1a), the virG gene may befurther inserted into a cosmid vector of the present inventioncontaining a nucleic acid fragment of a plant. In embodiments where acosmid vector of the present invention already carries the virG gene(e.g., pLC40GWHvG1), the virG gene need not be further inserted.

Alternatively, the virG gene may exist in an independent plasmidseparate from a cosmid vector of the present invention. In this case,Agrobacterium in the method of the present invention harbors a plasmidcapable of coexisting with an IncP plasmid in Agrobacterium cells andcontaining the virG gene of a bacterium of the genus Agrobacterium, inaddition to the cosmid vector (embodiment 1b).

The Ti plasmid or Ri plasmid is not specifically limited, but preferablydisarmed by deleting the T-DNA.

The plasmid containing the virG gene of a bacterium of the genusAgrobacterium in 1b) may contain an origin of replication of an IncWplasmid. Preferably, it is pVGW having the structure shown in FIG. 14.More preferably, it is pVGW2 having the structure shown in FIG. 15.

The plasmid containing the virG gene of a bacterium of the genusAgrobacterium in 1b) may further contain the virB gene of a bacterium ofthe genus Agrobacterium. Here again, the plasmid may contain an originof replication of an IncW plasmid. Such a plasmid is preferably pTOK47.

The virB gene of a bacterium of the genus Agrobacterium is described indetail in Ward et al. (1988) J Biol Chem 263: 5804-5814. For example, itcan be conventionally prepared from a plasmid such as pSB1 (Komari etal. 1996 Plant J 10: 165-174). The nucleotide sequence of virB isdefined as, e.g., nucleotides 3416-12851 of the nucleotide sequence ofGenbank/EMBL Accession Number: AB027255 (pSB1). As a non-limitativeexample, a DNA containing a nucleotide sequence hybridizing to thissequence or a complementary strand thereto under stringent conditionscan be used as the virB gene.

These transformation methods are more effective for plants normallyassociated with low efficiency of Agrobacterium-mediated transformation,e.g., including, but not limited to, maize and soybean. When the nucleicacid fragment to be transferred is large (e.g., 25-40 kb as anon-limitative example) or has a complex structure (e.g., a highlyrepeated sequence as a non-limitative example), pVGW described below ispreferably used as a plasmid containing the virG gene of a bacterium ofthe genus Agrobacterium.

Plasmid Vectors

In plants such as maize, wherein transformation is difficult to occur,the efficiency of Agrobacterium-mediated transformation with standardbinary vectors containing the T-DNA is very low except for special cases(Frame et al. 2002 Plant Physiol 129: 13-22). Previous reports show anincrease in the efficiency of transient expression in maize by thecoexistence of a binary vector with another plasmid containing thevirGN54D gene, a variant of the virG gene in Agrobacterium (Hansen etal. 1994 ProNAS 91:7603-7607), and a high efficiency maizetransformation system with a binary vector containing virG and virB(Ishida et al. 1996 Nat Biotechnol 14:745-50).

However, no report has shown that the maize transformation efficiencywas increased by the coexistence of a binary vector with a plasmidcontaining virG or virGN54D in Agrobacterium.

The cosmid vectors of the present invention (pLC vectors) (IncPplasmids) are also expected to further improve the maize transformationefficiency. Plasmids capable of coexisting with an IncP plasmid includee.g., IncW plasmids (Close et al. 1984 Plasmid 12: 111-118). Previouslyreported IncW vectors containing virG are large because they containorigins of replication of other plasmids such as pBR322 ori. Forexample, pTOK47 contains IncW (pSa) on and pBR322 on (as well as notonly virG but also virB) and it has a full length of about 28 kb (Jin etal. 1987 J Bacteriol 169: 4417-4425). pYW48 contains IncW (pSa) on andpBR322 on (as well as not only virG but also virA) and it has a fulllength of 15.5 kb (Wang et al. 2000 Gene 242: 105-114). Such vectors canalso be used in the transformation methods of the present invention.However, these vectors are so long that they may cause problems instability in bacteria when they coexist with a pLC vector containing alarge fragment, and therefore, small vectors capable of coexisting witha pLC vector and containing virG are desirable.

As a means to solve these problems, the present invention provides asmall plasmid vector capable of further improving the transformationefficiency by the coexistence with the cosmid vectors of the presentinvention described above.

The plasmid vector of the present invention satisfies all of thecriteria below.

1) it contains an origin of replication of an IncW plasmid, but does notcontain any origin of replication of other plasmid groups;

2) it contains the repA gene necessary for the replication of an IncWplasmid;

3) it contains a drug resistance gene expressed in E. coli and abacterium of the genus Agrobacterium; and

4) it contains the virG gene of a bacterium of the genus Agrobacterium.

1) The nucleotide sequence of the origin of replication of an IncWplasmid of the present invention is not specifically limited so far asit has the function as an origin of replication of an IncW plasmid.

The origin of replication of an IncW plasmid has molecular biologicalproperties described in detail in Okumura and Kado (1992 Mol Gen Genet235: 55-63), and it is defined as nucleotides 2170-2552 of Genbank/EMBLAccession Number: U30471 (full length 5500 bp). This corresponds tonucleotides 2832-3214 of SEQ ID NO: 8.

The origin of replication of an IncW plasmid can be conventionallyprepared from an IncW plasmid such as pTOK47 (Jin et al. 1987 JBacteriol 169: 4417-4425). For example, nucleotides 2832-3214 of SEQ IDNO: 8 in a 2.7 kb DNA amplified by PCR from pTOK47 with repA necessaryfor the replication of an IncW plasmid described below can be used.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 2832-3214 of SEQ ID NO: 8 described above under stringentconditions and having the function of an origin of replication of anIncW plasmid can also be used. Alternatively, a nucleic acid containinga nucleotide sequence having an identity of at least 95%, morepreferably 97%, still more preferably 99% to the nucleotide sequence ofnucleotides 2832-3214 of SEQ ID NO: 8 described above and having thefunction of an origin of replication of an IncW plasmid can also beused.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 2832-3214 of SEQ ID NO: 8 may be selected as a sequencehaving a similar function.

2) The nucleotide sequence of the repA gene of the present invention isnot specifically limited so far as it has the function as the repA genenecessary for the replication of an IncW plasmid.

The repA necessary for the replication of an IncW plasmid has molecularbiological properties described in detail in Okumura and Kado (1992 MolGen Genet 235: 55-63), and it is defined as nucleotides 1108-2079 ofGenbank/EMBL Accession Number:U30471 (full length 5500 bp). Thiscorresponds to nucleotides 1770-2741 of SEQ ID NO: 8.

The repA necessary for the replication of an IncW plasmid can beconventionally prepared from an IncW plasmid such as pTOK47 (Jin et al.1987 J Bacteriol 169: 4417-4425). For example, nucleotides 1770-2741 ofSEQ ID NO: 8 in a 2.7 kb DNA amplified by PCR from pTOK47 with an originof replication of an IncW plasmid described above can be used.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of the nucleotide sequence ofnucleotides 1770-2741 of SEQ ID NO: 8 described above under stringentconditions and having the function of the repA gene necessary for thereplication of an IncW plasmid can also be used. Alternatively, anucleic acid containing a nucleotide sequence having an identity of atleast 95%, more preferably 97%, still more preferably 99% to thenucleotide sequence of nucleotides 1770-2741 of SEQ ID NO: 8 describedabove and having the function of the repA gene necessary for thereplication of an IncW plasmid can also be used.

It will be recognized by those skilled in the art that a shorter regionin nucleotides 1770-2741 of SEQ ID NO: 8 may be selected as a sequencehaving a similar function.

3) The drug resistance gene expressed in E. coli and a bacterium of thegenus Agrobacterium is used as a selectable marker for transformation.This drug resistance gene confers e.g., antibiotic resistance orautotrophy, including, but not limited to, a kanamycin resistance gene,a spectinomycin resistance gene, an ampicillin resistance gene, atetracycline resistance gene, a gentamycin resistance gene, a hygromycinresistance gene, etc.

4) The virG gene of a bacterium of the genus Agrobacterium and virGN54Dhave molecular biological properties described in detail in Winans etal. (1986) Proc. Natl. Acad. Sci. USA 83: 8278-8282 and Pazour et al.(1992) J. Bacteriol. 174: 4169-4174, Hansen et al. 1994 Proc. Natl.Acad. Sci. USA 91: 7603-7607, respectively. virG is a transcriptionregulator (activator) of other vir genes such as virB and virE. virG isactivated upon regulation (phosphorylation) by virA, whereas virGN54D isa variant in a permanently activated state without this regulation. ThevirG gene can be prepared by conventional procedure and virGN54D can beprepared by mutagenesis both from a plasmid such as pTOK47 (Jin et al.1987 J Bacteriol 169: 4417-4425). For example, 1 kb virG DNA(nucleotides 4024-5069 of SEQ ID NO: 7) amplified by PCR from pTOK47 and1 kb virGN54D DNA (nucleotides 1-1080 of SEQ ID NO: 8) amplified andprepared by PCR mutagenesis can be used.

Alternatively, a nucleic acid containing a nucleotide sequencehybridizing to a complementary strand of these nucleotide sequencesunder stringent conditions and having the function of the virG gene of abacterium of the genus Agrobacterium or a nucleic acid containing anucleotide sequence having an identity of at least 95%, more preferably97%, still more preferably 99% to these nucleotide sequences and havingthe function of the virG gene of a bacterium of the genus Agrobacteriumcan also be used.

The plasmid vector of the present invention preferably has a full lengthof 10 kb or less, more preferably 5 kb or less.

The plasmid vector of the present invention is preferably the pVGWvector having the structure shown in FIG. 14. More preferably, it ispVGW2 having the structure shown in FIG. 15. pVGW and pVGW2 are vectorssatisfying all of criteria 1)-4) above. pVGW shown as SEQ ID NO: 8 has afull length of 4531 bp, and pVGW2 shown as SEQ ID NO: 67 has a fulllength of 4836 bp, and they are characterized in that:

1) they contain an origin of replication of an IncW plasmid, but do notcontain any origin of replication of other plasmid groups;

2) they contain the repA gene necessary for the replication of an IncWplasmid;

3) they contain a gentamycin resistance gene as a drug resistance geneexpressed in E. coli and a bacterium of the genus Agrobacterium; and

4) they contain the virGN54D gene of a bacterium of the genusAgrobacterium.

Among these components, the origin of replication of an IncW plasmid andthe repA gene necessary for the replication of an IncW plasmid weresimultaneously cloned and the gentamycin resistance gene and thevirGN54D gene were separately cloned, after which all of the three DNAfragments (four components) were assembled.

Those skilled in the art can readily derive equivalents to the twoplasmid vectors pVGW, pVGW2 of the present invention described above,said equivalents having similar functions to those of these vectors evenif the nucleotide sequences are not completely identical. Thus, these“equivalents” are also included as preferred embodiments of the plasmidvectors of the present invention.

For example, it is thought that even if the nucleotide sequences of theplasmid vectors of the present invention are modified especially inparts other than the elements related to criteria 1)-4) above (e.g., theorigin of replication of an IncW plasmid in criterion 1)), they performsimilar functions to those of the original vectors as plasmid vectors.Moreover, more than one genes having similar functions to those of thedrug resistance gene in 3) among criteria 1)-4) are known even if thenucleotide sequences are not completely identical to the nucleotidesequences in the plasmid vectors, and those skilled in the art canmodify these parts as appropriate.

Therefore, an “equivalent” to each of the plasmid vectors of the presentinvention preferably refers to a nucleotide sequence identical to orhaving an identity of at least 95% or more, 97% or more, 98% or more or99% or more, more preferably 99.5% or more to the nucleotide sequence ofeach plasmid vector in the nucleotide sequences of the elements relatedto criteria 1)-2) and 4) of the plasmid vectors of the present inventionor refers to a nucleotide sequence hybridizing to a complementary strandof the nucleotide sequence of each plasmid vector under stringentconditions, said equivalent containing a mutation elsewhere in thenucleotide sequence while having similar function and effect to those ofeach vector. More preferably, it refers to a nucleotide sequenceidentical to the nucleotide sequence of each plasmid vector in thenucleotide sequences of the elements related to criteria 1)-4) of theplasmid vectors of the present invention and containing a mutationelsewhere in the nucleotide sequence while having similar function andeffect to those of each vector.

The degree of mutation is not specifically limited, but the “equivalent”preferably consists of a nucleotide sequence hybridizing to acomplementary strand of the nucleotide sequence of each plasmid vectorunder stringent conditions. The number of nucleotides that can bemutated is more preferably one or more, still more preferably one to afew (e.g., to the extent at which a mutation can be introduced by knownsite-directed mutagenesis).

The “equivalent” also preferably consists of a nucleotide sequencehaving an identity of 95% or more, 97% or more, 98% or more or 99% ormore, more preferably 99.5% or more to a nucleotide sequence selectedfrom the nucleotide sequences of the plasmid vectors.

As used herein, the expression “under stringent conditions” refers tohybridization under conditions of moderate or high stringency.Specifically, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 3rd Ed.,Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001, and include useof a prewashing solution for the nitrocellulose filters containing5×SSC, 0.5% SDS, 1.0 raM EDTA (pH 8.0), hybridization conditions of2×SSC to 6×SSC with or without about 50% formamide at about 40° C. to50° C. (or other similar hybridization solution, such as Stark'ssolution, in about 50% formamide at about 42° C.), and washingconditions of 0.5 to 6×SSC, 0.1% SDS at about 40° C. to 60° C.Preferably, conditions of moderate stringency include hybridizationconditions (and washing conditions) of 6×SSC at about 50° C. Conditionsof high stringency can also be readily determined by the skilled artisanbased on, for example, the length of the DNA.

Generally, such conditions include hybridization and/or washing athigher temperatures and/or lower salt concentrations than in theconditions of moderate stringency (e.g., hybridization in 6×SSC to0.2×SSC, preferably 6×SSC, more preferably 2×SSC, most preferably0.2×SSC at about 65° C.), and are defined to involve hybridizationconditions as above and washing in 0.2×SSC, 0.1% SDS at about 65° C. to68° C. SSPE (1×SSPE=0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH7.4) can be substituted for SSC (1×SSC=0.15 M NaCl and 15 mM sodiumcitrate) for use as hybridization and washing buffers, and washing iscontinued for 15 minutes after completion of hybridization.

Commercially available hybridization kits not using radioactivesubstances as probes can also be used. Specifically, hybridization canbe performed by using ECL direct labeling & detection system (fromAmersham), etc. Stringent hybridization conditions include hybridizationin the hybridization buffer included in the kit containing 5% (w/v)Blocking reagent and 0.5 M NaCl at 42° C. for 4 hours, followed bywashing twice in 0.4% SDS, 0.5×SSC at 55° C. for 20 minutes, and once in2×SSC at room temperature for 5 minutes.

pVGW is characterized in that it is small and stable. Specifically, itis effective for improving the transformation efficiency by thecoexistence with pLC especially when large fragments are used and/orwhen maize is used as a host. It is also effective for improving theefficiency of transformation of maize or the like by the coexistencewith an ordinary vector other than pLC.

Effects of the Invention

The vectors (pLC vectors) of the present invention provide the followingadvantages that could not be achieved by known vectors:

-   -   they allow efficient cloning of DNA fragments of about 25-40 kb        in size, preferably 30-40 kb;    -   they are stably maintained in E. coli and Agrobacterium cells;    -   they can be efficiently introduced in Agrobacterium;    -   the copy number per cell in E. coli and Agrobacterium is 4-5;        and    -   they allow efficient transfer of only cloned DNA fragments of        interest into plants, preferably monocotyledons (the        transformation efficiency of pLC vectors is 90%, in contrast to        the transformation efficiency of pSB vectors 60%).

The combined use of the pLC vectors of the present invention and thepVGW vector allows efficient gene transfer into even plants that arerelatively difficult to transform such as maize.

Candidate gene sites can be narrowed down with little expenditure oflabor and time by map-based cloning with the pLC vectors of the presentinvention.

The present invention is significantly effective even if mappinginformation is very limited. For example, suppose that nothing is knownexcept for the presence of candidate genes at an end region of achromosome. If the entire length of one chromosome is 40 Mb, forexample, its end region may be assumed to be 2 Mb. This region can becovered by about 20 BAC clones carrying an insert fragment of 150 kb onaverage by constructing a library of aligned BACs (BAC contig).Therefore, if 20 subclones are prepared from each BAC, almost allcandidate genes can be rapidly identified by preparing a total of 400fragments and 4000 recombinants. Thus, genes can be identified withlittle expenditure of labor and time unimaginable from conventionaltechniques by using the technique of the present invention.

EMBODIMENTS OF THE PRESENT INVENTION

The present invention preferably the following embodiments.

Embodiment 1

A cosmid vector having a full length of 15 kb or less characterized inthat:

1) it contains an origin of replication (oriV) of an IncP plasmid, butdoes not contain any origin of replication of other plasmid groups;

2) it contains the trfA1 gene of an IncP plasmid;

3) it contains an origin of conjugative transfer (oriT) of an IncPplasmid;

4) it contains the incC1 gene of an IncP plasmid;

5) it contains a cos site of lambda phage and the cos site is locatedoutside the T-DNA;

6) it contains a drug resistance gene expressed in E. coli and abacterium of the genus Agrobacterium;

7) it contains a T-DNA right border sequence of a bacterium of the genusAgrobacterium;

8) it contains a T-DNA left border sequence of a bacterium of the genusAgrobacterium;

9) it contains a selectable marker gene for plant transformation locatedbetween 7) and 8) and expressed in a plant; and

10) it contains restriction endonuclease recognition site(s) locatedbetween 7) and 8) for cloning a foreign gene.

Embodiment 2

The cosmid vector of Embodiment 1 wherein the selectable marker gene forplant transformation is selected from the group consisting of ahygromycin resistance gene, a phosphinotricin resistance gene and akanamycin resistance gene.

Embodiment 3

The cosmid vector of Embodiment 1 or 2, which contains the korB gene ofan IncP plasmid.

Embodiment 4

The cosmid vector of any one of Embodiments 1 to 3 selected from thegroup consisting of:

the cosmid vector pLC40 consisting of the nucleotide sequence of SEQ IDNO: 2 or an equivalent thereof;

the cosmid vector pLC40GWH consisting of the nucleotide sequence of SEQID NO: 3 or an equivalent thereof;

the cosmid vector pLC40 bar consisting of the nucleotide sequence of SEQID NO: 4 or an equivalent thereof;

the cosmid vector pLC40GWB consisting of the nucleotide sequence of SEQID NO: 5 or an equivalent thereof;

the cosmid vector pLC40GWHKorB consisting of the nucleotide sequence ofSEQ ID NO: 65 or an equivalent thereof;

the cosmid vector pLCleo consisting of the nucleotide sequence of SEQ IDNO: 66 or an equivalent thereof; and

the cosmid vector pLC40GWHvG1 consisting of the nucleotide sequence ofSEQ ID NO: 7 or an equivalent thereof.

Embodiment 5

The cosmid vector of Embodiment 4 selected from the group consisting of:

the cosmid vector pLC40 consisting of the nucleotide sequence of SEQ IDNO: 2;

the cosmid vector pLC40GWH consisting of the nucleotide sequence of SEQID NO: 3;

the cosmid vector pLC40 bar consisting of the nucleotide sequence of SEQID NO: 4;

the cosmid vector pLC40GWB consisting of the nucleotide sequence of SEQID NO: 5;

the cosmid vector pLC40GWHKorB consisting of the nucleotide sequence ofSEQ ID NO: 65;

the cosmid vector pLCleo consisting of the nucleotide sequence of SEQ IDNO: 66; and

the cosmid vector pLC40GWHvG1 consisting of the nucleotide sequence ofSEQ ID NO: 7.

Embodiment 6

A method for transforming a plant, comprising transforming the plantwith a bacterium of the genus Agrobacterium harboring an expressionvector containing a nucleic acid fragment of a plant inserted into thecosmid vector of any one of Embodiments 1 to 5.

Embodiment 7

The method of Embodiment 6 wherein the nucleic acid fragment insertedhas a size of 25-40 kb.

Embodiment 8

The method of Embodiment 6 or 7 characterized in that it uses abacterium of the genus Agrobacterium harboring the following elementsfor transforming the plant:

1a) a vector containing a nucleic acid fragment of a plant and the virGgene of a bacterium of the genus Agrobacterium inserted into the cosmidvector of any one of Embodiments 1 to 5; or

1b) a vector containing a nucleic acid fragment of a plant inserted intothe cosmid vector of any one of Embodiments 1 to 5, and a plasmidcapable of coexisting with an IncP plasmid in a cell of a bacterium ofthe genus Agrobacterium and containing the virG gene of a bacterium ofthe genus Agrobacterium, and

2) a Ti plasmid or Ri plasmid of a bacterium of the genus Agrobacterium.

Embodiment 9

The method of Embodiment 8 wherein the virG gene of a bacterium of thegenus Agrobacterium in 1a) or 1b) is virGN54D.

Embodiment 10

The method of Embodiment 8 wherein the plasmid containing the virG geneof a bacterium of the genus Agrobacterium in 1b) contains an origin ofreplication of an IncW plasmid.

Embodiment 11

The method of Embodiment 10 wherein the plasmid containing the virG geneof a bacterium of the genus Agrobacterium in 1b) is pVGW having thestructure shown in FIG. 14 or pVGW2 having the structure shown in FIG.15.

Embodiment 12

The method of Embodiment 8 wherein the plasmid containing the virG geneof a bacterium of the genus Agrobacterium in 1b) further contains thevirB gene of a bacterium of the genus Agrobacterium.

Embodiment 13

The method of Embodiment 12 wherein the plasmid containing the virG geneof a bacterium of the genus Agrobacterium in 1b) contains an origin ofreplication of an IncW plasmid.

Embodiment 14

The method of Embodiment 13 wherein the plasmid containing the virG geneof a bacterium of the genus Agrobacterium in 1b) is pTOK47.

Embodiment 15

A map-based cloning method comprising the steps of:

1) partially or completely digesting BAC clones containing candidategenes responsible for a plant phenotype with a restriction endonuclease;

2) subcloning DNA fragments obtained in step 1) using a cosmid vector toconstruct a library; and 3) individually transferring clonesconstituting the library into a plant to evaluate the phenotypes oftransformed plants.

Embodiment 16

The map-based cloning method of Embodiment 15 wherein the DNA fragmentsobtained in step 1) have a size of 25-40 kb.

Embodiment 17

The map-based cloning method of Embodiment 16 wherein the cosmid vectorin 2) is the cosmid vector of any one of Embodiments 1 to 5.

Embodiment 18

A plasmid vector characterized in that:

1) it contains an element necessary for the replication of an IncWplasmid, but does not contain any origin of replication of other plasmidgroups;

2) it contains the repA gene necessary for the replication of an IncWplasmid;

3) it contains a drug resistance gene expressed in E. coli and abacterium of the genus Agrobacterium; and

4) the virG gene of a bacterium of the genus Agrobacterium.

Embodiment 19

The plasmid vector of Embodiment 18, which has a full length of 10 kb orless.

Embodiment 20

The plasmid vector of Embodiment 19 wherein the virG gene of a bacteriumof the genus Agrobacterium is virGN54D.

Embodiment 21

The plasmid vector of any one of Embodiments 18 to 20 selected from thegroup consisting of:

the plasmid vector pVGW consisting of the nucleotide sequence of SEQ IDNO: 8 or an equivalent thereof; and

the plasmid vector pVGW2 consisting of the nucleotide sequence of SEQ IDNO: 67 or an equivalent thereof.

Embodiment 22

The plasmid vector pVGW consisting of the nucleotide sequence of SEQ IDNO: 8.

Embodiment 23

The plasmid vector pVGW2 consisting of the nucleotide sequence of SEQ IDNO: 67.

Embodiment 24

A method for transforming a plant, comprising transforming the plantwith a bacterium of the genus Agrobacterium harboring the plasmid vectorof any one of Embodiments 18 to 23.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the vector pSB200PcHm.

FIG. 2 is a schematic diagram of the vector pSB25UNpHm.

FIG. 3 is a schematic diagram of the vector pSB200PcHmGWH.

FIG. 4 is a schematic diagram of the vector pSB200PcHmGWB.

FIG. 5 is a diagram showing a procedure for constructing the vectorpLC40.

FIG. 6 is a schematic diagram of the vector pLC40.

FIG. 7 is a schematic diagram of the vector pLC40GWH.

FIG. 8 is a schematic diagram of the vector pLC40 bar.

FIG. 9 is a schematic diagram of the vector pLC40GWB.

FIG. 10 is a schematic diagram of the vector pLC40GWHkorB.

FIG. 11 is a schematic diagram of the vector pLCleo.

FIG. 12 is a schematic diagram of the vector pLCSBGWBSWa.

FIG. 13 is a schematic diagram of the vector pLC40GWHvG1.

FIG. 14 is a schematic diagram of the vector pVGW.

FIG. 15 is a schematic diagram of the vector pVGW2.

FIG. 16 shows the results of cloning of a genomic DNA fragment by a pLCvector. An example of a teosinte genomic DNA fragment is shown. M1:marker (1 kb ladder), M2: marker (X-HindIII); the numbers representclone numbers, and the arrow indicates the size of the bandcorresponding to pLC40GWH (13.2 kb). Plasmid DNA of eleven clones from ateosinte library was purified. The DNA was cleaved with the restrictionendonucleases HindIII and SacI in the multicloning site at each end ofthe plasmid insert and separated by agarose gel (0.8%) electrophoresis.

FIG. 17 shows the results of transformation of a genomic DNA fragmentinto rice (in the center region of fragment B). M: markers, Yu:Yukihikari, Ru: Oryza rufipogon, Transgenic: transformed rice (twoindividuals). In the transformed rice, a band derived from Oryzarufipogon was detected in addition to a band derived from Yukihikari.

EXAMPLES

The following examples further illustrate the present invention but arenot intended to limit the technical scope of the invention. Thoseskilled in the art can readily add modifications/changes to the presentinvention in the light of the description herein, and thosemodifications/changes are also included in the technical scope of thepresent invention.

Example 1 Construction of pLC Series Cosmid Vectors

In the following procedures, molecular biological experimental methodswere performed as described in Sambrook J. and Russell D. W. 2001.Molecular Cloning, A Laboratory Manual, 3rd edn. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA., unless otherwisespecified.

1) Construction of T-DNA Regions

A PacI linker (gttaattaac) (SEQ ID NO: 10) was inserted into the EcoRVsite of pSB200 (WO2005/040374) to construct pSB200Pac. The cauliflowermosaic virus ³⁵S promoter in pSB25 (Ishida et al. 1996) was replaced bythe ubiquitin promoter of maize (Christensen et al. 1992 Plant Mol Biol18: 675-689) to construct pSB25U. The adapters HinNspISceRV andHinNspISceFW (Table 1) having recognition sites for the restrictionendonuclease NspV and the homing endonuclease I-SceI were annealed. Apart of the annealed adapters were phosphorylated with a polynucleotidekinase (PNK, Amersham). The phosphorylated adapters were cloned into theSacI site of pSB200Pac and the HindIII site of pSB25U. The resultingplasmids were designated as pSB200PacHm1 and pSB25UNpHm1, respectively.

The adapters SpeICeuRV and SpeICeuFW containing a homing endonucleaseI-CeuI site (Table 1) were inserted into the SpeI site of pSB200PacHm1and pSB25UNpHm1. This operation generated the vector pSB200PcHmcontaining homing endonuclease sites I-SceI and I-CeuI inserted into theSacI and SpeI sites respectively of pSB200Pac (FIG. 1), and the vectorpSB25UNpHm containing homing endonuclease sites I-SceI (+NspV site) andI-CeuI inserted into the HindIII and SpeI sites respectively of pSB25U(FIG. 2). Excision by I-SceI and I-CeuI was verified, and the nucleotidesequences were checked by using ABI PRISM Fluorescent Sequencer (Model310 Genetic Analyzer, from Perkin Elmer) to confirm that a singleadapter had been inserted. In these vectors, the I-SceI-selectablemarker unit-LB-1-CeuI can be excised.

TABLE 1 Primer Name Sequence Length HinNspISceRV5′-AgC TTT CgA ATA ggg ATA ACA ggg TAA T-3′ 28 mer HinNspISceFW5′-AgC TAT TAC CCT gTT ATC CCT ATT CgA A-3′ 28 mer SpeICeuRV5′-CTA gTA ACT ATA ACg gTC CTA Agg TAg CgA C-3′ 31 mer SpeICeuFW5′-CTA ggT CgC TAC CTT Agg ACC gTT ATA gTT A-3′ 31 mer SEQ ID NOs: 23-26in order from the top.

Then, pSB200PcHm was digested with BamHI to remove the hygromycinresistance gene (hpt), and then blunt-ended. This was ligated to theaatR1-ccdB-Cm-aatR fragment (Invitrogen), and transferred into E. coliDB3.1 to select a chloramphenicol-resistant colony, thereby generatingthe destination vector pDEST3342. Then, the following primers containingan aatB sequence were synthesized (aatB sequences are shown inuppercase) in order to introduce a marker gene into the pDONR/Zeoplasmid (Invitrogen) by the BP reaction.

TABLE 2 Primer Name Sequence Length aatB1-HPTggg gAC AAG TTT GTA CAA AAA AGC AGG CTc aat gag ata tga aaa agc c 49 merHPT-aatB2ggg gAC CAC TTT GTA CAA GAA AGC TGG GTc tat tcc ttt gcc ctc gga cga g52 mer aatB1-barggg gAC AAG TTT GTA CAA AAA AGC AGG CTc cat gga ccc aga acg acg c 49 merbar-aatB2ggg gAC CAC TTT GTA CAA GAA AGC TGG GTt cct aga cgc gtg aga tca g 49 merSEQ ID NOs: 27-30 in order from the top.

For the amplification of the Hpt gene, the hpt gene described in Bilanget al. (1991) Gene 100: 247-250 was used as a template DNA along withaatB1-HPT and HPT-aatB2 as primers. For the amplification of thephosphinotricin resistance gene (bar), pSB25 (Ishida et al. 1996) wasused as a template DNA along with aatB1-bar and bar-aatB2 as primers(Table 2). In 100 μl of a reaction solution containing 10 ng of eachtemplate DNA and 25 pmoles of the primers, 35 cycles of PCR wasperformed. After the completion of the reaction, the products wererecovered by ethanol precipitation and used for the BP reaction (25° C.,6 hrs) according to the protocol attached to BP Clonase Enzyme Mix kit(Invitrogen), and then transferred into E. coli DH5a and E. coli cellsharboring plasmids of interest were selected on a low salt LA platecontaining the antibiotics Zeocin. The nucleotide sequences of thefinally obtained plasmids were confirmed by restriction endonucleaseanalysis, thereby generating pENT-HPTwt and pENT-bar, respectively.

The destination vector (pDEST3342) prepared before and the entry vectors(pENT-HPTwt and pENT-bar) were used to prepare final plasmids ofinterest by the LR reaction. After the reaction in 20 μl of a reactionsolution (containing 300 ng each of the destination vector and entryvectors) at 25° C. for 4 hours according to the protocol attached toGATEWAY LR Clonase Enzyme Mix, the reaction products were transferredinto E. coli DH5a by electroporation. Plasmid DNAs were prepared fromcolonies grown on an LA plate containing spectinomycin and candidateclones were selected by restriction fragment patterns. They wereconfirmed by nucleotide sequence analysis to contain an aatB sequenceand the sequence of the HPT gene or bar gene and designated aspSB200PcHmGWH (FIG. 3) and pSB200PcHmGWB (FIG. 4), respectively.

2) Construction of the Cosmid Vector pLC40

A PCR reaction was performed using Pyrobest DNA Polymerase (Takara)along with OriV3′ClaFW, OriV5′PvNhEc, OriT5′Bg1RV, OriT3′SpEcFW,InC5′XbRV, InC3′BgEcFW, R5′XhoIRV, R3′BmEcFW, 121KIII5′NspV,121KIII3′SalI, COS5′BmRV and COS3′MunFW designed as PCR primers foramplifying a DNA fragment containing oriV, a DNA fragment containingoriT, a DNA fragment containing the incC2 gene, a DNA fragmentcontaining the trfA1 gene, all of which are derived from the IncPplasmid pVK102 (Knauf and Nester, Plasmid 8: 45-54, 1982), a DNAfragment containing the nptIII gene from pBI121 and a DNA fragmentcontaining cos from pSB11 (Table 3).

Each primer contains a restriction endonuclease site for later use. ThePCR products other than the trfA1 gene, i.e., the DNA fragmentcontaining oriV from pVK102 (884 bp), the DNA fragment containing oriT(810 bp), the DNA fragment containing the incC1 gene (2118 bp), the DNAfragment containing the nptIII gene from pBI121 (1087 bp), and the DNAfragment containing cos from pSB11 were each cloned into the vectorpCR2.1Topo Blunt (from Invitrogen). As a result, the DNA fragmentcontaining oriV was found to contain two nucleotide substitutions andone nucleotide addition as compared with the corresponding nucleotidesequence in a public database (Genbank accession L27758). Thesemutations were also found in the template plasmid, showing that theywere not introduced by PCR but that the template plasmid had anucleotide sequence different from the sequence in the public database.The nucleotide sequences of oriT, the incC1 gene, and cos werecompletely identical to those in the database. However, the trfA1 genecould not be cloned alone. Thus, the construction was pursued by themethod described below.

The plasmid into which the DNA fragment containing oriV had been clonedwas digested with the restriction endonucleases EcoRI and ClaI, and a0.9 kb fragment was purified. Similarly, the DNA fragment containingoriT was digested with EcoRI and BglII, and the DNA fragment containingnptIII was digested with NspV and SalI, and the digests were purified.The PCR product of the DNA fragment containing the trfA1 gene wasprecipitated with ethanol, and then digested with XhoI and BamHI, andpurified. These 4 fragments (oriV, the trfA1 gene, nptIII, oriT) wereligated at a time and cloned together. The nucleotide sequence of theresulting plasmid (designated as pVRKT) was analyzed to reveal aframeshift mutation in the DNA fragment containing the trfA1 gene ascompared with the corresponding nucleotide sequence in the publicdatabase (Genbank accession L27758), but the same mutation was alsofound in pVK102 used as the template, thereby concluding that themutation was not introduced by PCR and that pVK102 used as the templatecontained a nucleotide sequence different from the sequence in thepublic database.

The resulting plasmid pVRKT containing the 4 fragments were digestedwith EcoRI and SpeI, and the DNA fragment containing the incC1 generecovered by digesting the plasmid containing the incC1 gene with EcoRIand XbaI was inserted into it. The resulting plasmid was furtherdigested with EcoRI and BglII, and the DNA fragment containing cosrecovered by digesting the plasmid containing the DNA fragmentcontaining cos with MunI and BamHI was inserted into it to generate thelow-copy vector backbone p6FRG (about 8.5 kb) consisting of the 6fragments, i.e., the DNA fragment containing oriV, the DNA fragmentcontaining the trfA1 gene, the DNA fragment containing nptIII, the DNAfragment containing oriT, the DNA fragment containing the incC1 gene,and the DNA fragment containing cos(SEQ ID NO: 1 in the SequenceListing). The foregoing cloning procedure was summarized in a schematicdiagram shown in FIG. 5. The T-DNA region (SspI-SpeI fragment) ofpSB200PcHm was inserted into the PvuII, NheI sites of the p6FRG plasmidto generate the vector pLC40 (FIG. 6, SEQ ID NO: 2 in the SequenceListing).

TABLE 3 Primer Name Sequence Target gene Length 121KIII5′NspV5′-TCg TTC gAA TCg ATA CTA TgT TAT ACg CCA AC-3′ nptIII 32 mer121KIII3′SalI 5′-ATC gTC gAC TgC ACg AAT ACC AgC gAC CC-3′ 29 merCOS5′BmRV 5′-ggg ggA TCC TTC CAT TgT TCA TTC CAC ggA C-3′ cos 31 merCOS3′MunFW 5′-ggg CAA TTg ACA TgA ggT TgC CCC gTA TTC -3′ 30 merOriV3′ClaFW 5′-gAT ATC gAT AgC gTg gAC TCA Agg CTC TC-3′ oriV 29 merOriV5′PvNhEc5′-AAA gAA TTC gCT AgC CAg CTg gCg CTg CCA TTT TTg ggg Tg-3′ 41 merR5′XhoIRV 5′-AAA CTC gAg CAg CCg AgA ACA TTg gTT CC-3′ trfA1 29 merR3′BmEcFW 5′-TAg gAA TTC ggA TCC AAA ACA ACT gTC AAA gCg CAC-3′ 36 merOriT5′BglRV 5′-CgT AgA TCT ggC gCT Cgg TCT TgC CTT g-3′ oriT 28 merOriT3′SpEcFW 5′-TgT gAA TTC ACT AgT gAT ATT CCA CAA AAC AgC Agg g-3′37 mer InC5′XbRV 5′-CCg TCT AgA TTC gAg CCA Cgg Tag Cgg C-3′ incC228 mer InC3′BgEcFW 5′-CTT gAA TTC AgA TCT TCT Cgg Cgg CgA TCA CgA C-3′34 mer SEQ ID NOs: 31-42 in order from the top.

3) Construction of Other pLC Series Cosmid Vectors pLC40GWH

Of the two BalI sites in the backbone of pSB200PcHmGWH, the one on theleft side of the RB is not cleaved because it is methylated. Thus,pSB200PcHmGWH was used in the experiments below after it was oncetransferred into the E. coli strain GM48 to demethylate that site.pSB200PcHmGWH was treated with BalI and SpeI to excise a regioncontaining the T-DNA, which was cloned into the PvuII, NheI sites of6FRG described above to generate pLC40GWH (FIG. 7, SEQ ID NO: 3 in theSequence Listing). This differs from pLC40 by insertions of attB1, 2sequences and a deletion of a 317 by SspI-BalI region upstream of theRB.

pLC40 bar, pLC40GWB, pLC40GWBSW

pSB25UNpHm and pSB200PcHmGWB were digested with the restrictionendonucleases SpeI and SspI, and fragments containing the T-DNA wererecovered. These fragments were cloned into the PvuII, NheI sites ofp6FRG to generate pLC40 bar (FIG. 8, SEQ ID NO: 4 in the SequenceListing) and pLC40GWB (FIG. 9, SEQ ID NO: 5 in the Sequence Listing),respectively. pSB200PcHmGWB was treated with NspV, blunt-ended, anddephosphorylated. A pSwaI linker (Table 4) was inserted into this site(pSB200PcHmGWBSW). This plasmid was digested with the restrictionendonucleases SpeI and SspI, and a fragment containing the T-DNA wasrecovered. These fragments were cloned into the PvuII, NheI sites ofp6FRG to generate pLC40GWBSW.

pLC40:35S-IGUS, pLC40GWB:35S-IGUS

The vector pSB24 (Komari et al. 1996) was treated with the restrictionendonucleases HindIII and EcoRI to excise a DNA fragment consisting of³⁵S promoter-1-GUS gene-NOS terminator. This fragment was furtherblunt-ended by Klenow treatment, and then a 3.1 kb fragment was purifiedand recovered. The cosmid vector pLC40 described above was treated withthe restriction endonuclease NspV, blunt-ended with Klenow enzyme, andthen dephosphorylated and purified. On the other hand, pLC40GWBSW wastreated with the restriction endonuclease SwaI, dephosphorylated andthen gel-purified. The DNA fragment containing the GUS gene describedabove was inserted into these vectors to prepare pLC40:35S-IGUS andpLC40GWB:35S-IGUS, respectively.

pLC40GWHKorB

The cloned region of IncC1 in the pLC vector was extended, and thevector pLC40GWHKorB containing the korB gene was constructed.IncC3′BgEcFw (described above) and IncC/KorB-Xba#1 (Table 4) weredesigned as primers for amplifying a DNA fragment containing IncC1-KorBof the IncP-based plasmid pVK102. Each primer contains a restrictionendonuclease site for later use. A PCR reaction was performed asfollows. In 50 μl of a reaction solution containing 500 ng of the pVK102plasmid DNA, 5 μl of 10× Pyrobest Buffer II, 4 μl of 2.5 mM each dNTP,50 pmoles of the primers, and 0.5 μl of Pyrobest DNA Polymerase (fromTakara), one cycle of 96° C. for 3 minutes, and 10 cycles of 96° C. for1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes and 30 secondswere performed by using Mastercycler gradient (eppendorf). The resultingamplified PCR product of IncC1-korB (3065 bp) was cloned into the vectorpCR2.1Topo Blunt (from Invitrogen). Ligation reactions were performedfollowing the instructions attached to the vector kit. The DNA wastransferred into E. coli DH5a by electroporation, and incubatedovernight at 37° C. on a 2×YT agar plate containing the antibioticZeocin (25 μg/ml). Colony direct PCR was performed to select candidateclones by using grown colonies as templates along with the same primerset as used for the amplification of IncC1-KorB. PCR conditions includedone cycle of 96° C. for 3 minutes, and 30 cycles of 96° C. for 1minutes, 55° C. for 1 minute, and 72° C. for 2 minutes and 30 secondsusing Wastercycler gradient in a suspension of the colony in 20 μl of areaction solution containing 2 μl of 10× Extaq Buffer, 1.6 μl of 2.5 mMeach dNTP, 5 pmoles of the primers, and 0.4 μl of Extaq DNA Polymerase(from Takara). The resulting PCR amplified products of about 3 kb wereselected as candidate clones. The nucleotide sequences of these cloneswere determined by ABI PRISM Fluorescent Sequencer (Model 3100 GeneticAnalyzer, from Applied Biosystems). As a result, the nucleotide sequenceof IncC1-KorB was completely identical to the sequence in the database.

Then, the plasmid pVRKT described above was digested with EcoRI andSpeI, and the IncC1-KorB fragment recovered by digesting the plasmidcontaining IncC1-KorB with EcoRI and XbaI was inserted into it. Theresulting plasmid was further digested with EcoRI and BglII, and the cosfragment (MunI-BamHI fragment) described above was inserted into it togenerate the plasmid p6FRG2 consisting of the 6 fragments, i.e., oriV,trfA1, nptIII, oriT, IncC1-KorB and cos. The T-DNA region (Ball-SpeIfragment) from pSB3342GWH was inserted into the PvuII, NheI sites of thep6FRG2 plasmid to generate the vector pLC40GWHKorB (FIG. 10, SEQ ID NO:65).

pLC40GWHKorBPI

In order that the cloned large genomic fragment could be excised in itsintact form, a recognition site for the homing endonuclease PI-SceI wasadded upstream of the multicloning site. pLC40GWHKorB was digested withHindIII, and PI-SceI adapters (PI-SceIFw, PI-SceIRv, Table 4) wereinserted to generate pLC40GWHKorBPI.

pLC40GWHKorBPIattB3

In order that the promoter of the selectable marker of pLC40GWH could bechanged by any other one by a Gateway system, an attB3 site was addedupstream of the ubiquitin promoter. pLC40GWHKorBPI was digested withI-SceI and attB3 adapters (attB3Fw, attB3Rv, Table 4) were inserted toprepare pLC40GWHKorBPIattB3.

pLCleo

In order that an NotI-digested genomic fragment could be cloned, arecognition site for PspOMI (producing the same sticky end as that ofNotI) was formed at the multicloning site and simultaneously therecognition site of ApaI (a neoschizomer of PspOMI) in the ubiquitinintron was abolished. pLC40GWHKorBPIattB3 was digested with ApaI andNheI, and ApaIm-NheI adapters (Apalm-NheIFw, Apalm-NheIRv, Table 4) wereinserted to prepare pLC40GWHKorBPIattB3ApaIm. This plasmid was digestedwith HindIII and NspV, and HindIII-PspOMI-NspV adapters(HindIII-PspOMI-NspVFw, HindIII-PspOMI-NspVRv, Table 4) were inserted tofinally prepare pLCleo (FIG. 11, SEQ ID NO: 66 in the Sequence Listing).

TABLE 4 Primer/Adapter name Sequence(5′-3′) Length IncC/KorB-Xba#1CGG TCT AGA GTG CGC AGC AGC TCG TTA TC 29 mer PI-SceIFwAGC TAT CTA TGT CGG GTG CGG AGA AAG AGG TAA TGA AAT GGC A 43 merPI-SceIRv AGC TTG CCA TTT CAT TAC CTC TTT CTC CGC ACC CGA CAT AGA T43 mer attB3Fw CAG GGT AAT CAA CTT TGT ATA ATA AAG TTG ATA A 34 merattB3Rv CAA CTT TAT TAT ACA AAG TTG ATT ACC CTG TTA T 34 merApalm-NheIFwGGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTG 60 merApalnn-NheIRyCTAGCGCCGGATCTAACACAAACACGGATCTAACACAAACATGAACAGAAGTAGAACTACCCGGCC66 mer HindIII-PspOMI-NspVFw AGC TTG GGC CCT T 13 merHindIII-PspOMI-NspVRv AGG GCC CA 8 mer SEQ ID NOs: 68-76 in order fromthe top.

p6FRGSwKp

p6FRG was treated with PvuII and dephosphorylated. The adapterSwaIKpnIRV, SwaIKpnIFW (Table 5) DNAs having recognition sites for SwaIand KpnI were annealed. A part of this was phosphorylated with PNK(Amersham). This SwaI-KpnI linker was inserted into the PvuII site ofp6FRG to generate p6FRGSwKp. The KpnI site was designed for cloning aDNA fragment containing the virB gene and the virG gene derived from theAgrobacterium strain A281 in the next step, and the SwaI site wasdesigned for cloning the T-DNA in the step after next.

TABLE 5 Linker/Adapter name Sequence Length pSwaI linker5′-cca ttt aaa tgg-3′ 12 mer SwaIKpnIRV 5′-cca ttt aaa tgg tac cgg-3′18 mer SwaIKpnIFW 5′-ccg gta cca ttt aaa tgg-3′ 18 mer SEQ ID NOs: 43-45in order from the top.

p6FRGSVR, p6FRGSVRF

The vector pSB1 (Komari et al. 1996) was digested with KpnI, and a 14.8kb DNA fragment containing the virB gene and the virG gene wasrecovered. This fragment was inserted into the KpnI-treated anddephosphorylated vector p6FRGSwKp, thereby generating p6FRGSVR andp6FRGSVF.

pLCSBGWBSW

pSB200PcHmGWBSW was digested with SpeI and SspI, and a DNA fragmentcontaining the T-DNA region was blunt-ended with Klenow enzyme. Thisfragment was inserted into the SwaI-digested and dephosphorylated vectorp6FRGSVR, thereby generating pLCSBGWBSW (FIG. 12, SEQ ID NO: 6 in theSequence Listing). This vector is a low-copy vector having a full lengthof about 28 kb, which contains the virB gene and the virG gene derivedfrom the Agrobacterium strain A281 so that it may be used fortransformation of maize. It also contains a cos site, which allows easycloning of about 10-20 kb of DNA by a packaging reaction.

4) A pLC Vector Containing virG

The vector pLC40GWHvG containing the virG gene in the pLC40GWH vectorwas constructed by the procedure described below as a means forimproving the efficiency of plant transformation with a pLC40 seriescosmid vector.

Preparation of the virG Gene

The primers virGProSm and virGTerSm for amplifying the virG gene(including its promoter, the structural gene and the 3′ region) weredesigned and synthesized. These primers, and pTOK47 (Jin et al. 1987 JBacteriol 169: 4417-4425) as a template DNA, were used to amplify thevirG gene by PCR. As a result, the PCR product of about 1 kb wasamplified. A part of the product was cloned into the vector pCR2.1Topo(from Invitrogen) in the same manner as described above, and thenucleotide sequence was determined. The DNA sequence of the VirG genecontains an NspV site. This restriction site will be used as a cloningsite in a future vector. Thus, this site was removed by PCR mutagenesis.The first adenine in the NspV site (ttcgaa) was changed to guanine(ttcgga) to design and synthesize the primer virGonNspVRV and itscomplementary sequence virGonNspVFW. PCR was performed with two primersets, i.e., one consisting of VirGonNspVFW and the primer virGProSpeplaced upstream of the virG gene promoter and the other consisting ofvirGonNspVRV and the primer virGTerSpe placed downstream of the virGgene terminator. The virG gene cloned into pCR2.1Topo was used as atemplate. As a result, the product of about 400 by and the product ofabout 600 by were amplified by the former and latter sets, respectively.These products were purified and used as templates for the next PCRreaction. A PCR reaction was performed with the purified two PCRproducts as templates and the previous primers virGProSpe andvirGTerSpe. As a result, the PCR product of about 1 kb was amplified.The PCR product was cloned into the pCR2.1Topo vector, and thenucleotide sequence was determined to confirm the mutation(ttcgaa→ttcgga).

Similarly, the unmutated virG gene was amplified by PCR with virGProSpeand virGTerSpe, and cloned into pCR2.1Topo, and the nucleotide sequencewas determined. The primers used in PCR are summarized in Table 6.

TABLE 6 Designation Sequence 5′-3′ Length virGProSmTCA ATA CCC ggg gTA ACC TCg AAg CgT TTC AC 32 mer virGTerSmTgg TgA CCC ggg ACC TAT Cgg AAC CCC TCA C 31 mer virGProSpeTCA ATA ACT AgT gTA ACC TCg AAg CgT TTC AC 32 mer virGTerSpeTgg TgA ACT AgT ACC TAT Cgg AAC CCC TCA C 31 mer virGonNspVRVCTT gAg ATC gTT Cgg AAT CTg 21 mer virGonNspVFWCAg ATT CCg AAC gAT CTC AAg 21 mer SEQ ID NOs: 46-51 in order from thetop.

pLC40GWHvG1, pLC40GWHvGC1

The vector pLC40GWH was digested with the restriction endonucleasePvuII, and dephosphorylated. An SpeI linker (GACTAGTC, from Takara) wasinserted to prepare pLC40GWHSpe. This plasmid was digested with therestriction endonuclease SpeI and dephosphorylated. A fragment of about1 kb of the mutated virG gene excised with SpeI from the vector wasinserted into this plasmid to prepare pLC40GWHvG1 (FIG. 13, SEQ ID NO: 7in the Sequence Listing). Similarly, the unmutated virG gene wasinserted into pLC40GWHSpe to prepare pLC40GWHvGC1.

pLC40GWHvG1:35S-IGUS, pLC40GWHvGC1:35S-IGUS

In the same manner as described above, the vector pSB24 (Komari et al.1996) was treated with the restriction endonucleases HindIII and EcoRIto excise a DNA fragment containing the GUS gene, which was cloned intoa vector having a multicloning site SgfI-HindIII-EcoRI-SgfI. Theresulting plasmid was digested with SgfI to recover the DNA fragmentcontaining the GUS gene. At this point, both ends of the DNA fragmentcontaining the GUS gene are SgfI sites. The cosmid vector pLC40GWHvG1described above was treated with the restriction endonuclease PacI anddephosphorylated. The 3.1 kb SgfI fragment (the DNA fragment containingthe GUS gene) was cloned into it to generate pLC40GWHvG1:35S-IGUS.Similarly, 35S-IGUS-NOS was introduced into pLC40GWHvGC1 to preparepLC40GWHvGC1:35S-IGUS.

5) virG-Containing Vectors Capable of Coexisting with pLC

pVGW

pTOK47 is a large IncW plasmid of about 28 kb containing virG and virB(Jin et al. 1987 J Bacteriol 169: 4417-4425). Thus, a smaller vectorcapable of coexisting with a pLC vector and containing the origin ofreplication IncW ori, the virG gene, and a selectable marker gene(designated as pVGW) was designed and constructed.

The primers pSa5′EcT22 and pSa3′BglII for amplifying a fragmentcontaining IncW on from pTOK47 (Jin et al. 1987 J Bacteriol 169:4417-4425), and the primers Gm5′Bm and Gm3′Xh-2nd for amplifying thegentamycin resistance gene (gentamycin acetyltransferase) from pPH1JI(Hirsch and Beringer 1984 Plasmid 12: 139-141) were designed (Table 7).Each primer contains a restriction endonuclease site for later use.pTOK47 and pPH1JI were used as templates, respectively. Pyrobest DNAPolymerase (from TaKaRa) was used to perform PCR. As a result, a DNAfragment of about 2.7 kb containing IncW on and a DNA fragment of about0.7 kb corresponding to the gentamycin resistance gene were amplified.

On the other hand, the primer virGN54DFW for changing the amino acidresidue at position 54 of virG derived from pTOK47 from N to D by PCRmutagenesis (virGN54D, Hansen et al. 1994 Proc. Natl. Acad. Sci. USA 91:7603-7607), and its complementary sequence virGN54DRV were designed. PCRwas performed with two primer sets, i.e., one consisting of virGN54DFWand the primer virGProSal placed on the 5′ of the virG gene promoter andthe other consisting of virGN54DRV and the primer virGTerPst placed onthe 3′ of the virG gene terminator (Table 7). The pTOK47 plasmid wasused as a template. As a result, the product of about 0.4 kb and theproduct of about 0.7 kb were amplified by the former and latter sets,respectively. These products were purified and used as templates alongwith the previous primers virGProSal and virGTerPst to further perform aPCR reaction. As a result, the product (virGN54D) of about 1.1 kb wasamplified.

The PCR products of the fragment containing IncW ori, the gentamycinresistance gene, and virGN54D were cloned into the pCR-Blunt II-TOPOvector (Invitrogen). The nucleotide sequence was determined and comparedwith a publicly available sequence (Genbank/EMBL Accession Number:U30471) to reveal a deletion of 6 nucleotides in the fragment containingIncW ori, which was also found in pTOK47 used as a template. However,the nucleotide sequence of the gentamycin resistance gene was completelyidentical to the sequence in the database. virGN54D was found to containthe mutation at the desired site.

The plasmid into which the fragment containing IncW on had been clonedwas digested with EcoT22I and BglII, and a 2.7 kb fragment wasrecovered. Similarly, the gentamycin resistance gene was digested withBamHI and XhoI, and virGN54D was digested with SalI and PstI, and eachfragment was purified. These three fragments were ligated together(BglII and BamHI, XhoI and SalI, and PstI and EcoT22I produce the samesticky ends) to generate pVGW (FIG. 14, SEQ ID NO: 8 in the SequenceListing).

TABLE 7 Designation Sequence Length pSa5′EcT225′-aaa atg cat ggc atg ttt aac aga atc tg-3′ 29 mer pSa3′BglII5′-ttt aga tct act cgt tcg cgg agc tgg-3′ 27 mer Gm5′Bm5′-aaa gga tcc ttc atg get tgt tat gac tg-3′ 29 mer Gm3′Xh-2^(nd)5′-tgc ctc gag aca att tac cga aca act ccg-3′ 30 mer virGN54DFW5′-cga cct aaa tct aga tca aca ac-3′ 23 mer viGN54DRV5′-gtt gtt gat cta gat tta ggt cg-3′ 23 mer virGProSal5′-ttt gtc gac cat agg cga tct cct taa tc-3′ 29 mer virGTerPst5′-aaa ctg cag gtg aag agg gac cta tcg g-3′ 28 mer SEQ ID NOs: 52-59 inorder from the top.

pVGW2

To further increase the convenience of pVGW, the promoter region of thegentamycin resistance gene was extended and additional cloning siteswere added to construct the vector pVGW2. The primers BamSmaGmPro andNheIsiteGmRv for amplifying the gentamycin resistance gene of theplasmid pPH1JI, and the primers ‘MscIsite-virG5’ Fw (for these primers,see Table 8) and pSa3′BglII (described above) for amplifying thevirG-IncW region of pVGW were designed. Each primer contains arestriction endonuclease site. A PCR reaction was performed as follows.One cycle of 98° C. for 30 seconds and 35 cycles of 98° C. for 10seconds, 55° C. for 5 seconds, and 72° C. for 1 minute were performedusing Mastercycler gradient (eppendorf) in 50 μl of a reaction solutioncontaining 1 ng of the template plasmid DNA, 25 μl of 2× PrimeSTAR MaxPremix (from Takara), and 15 pmoles of the primers. As a result, the PCRproducts of the gentamycin resistance gene (826 bp) and the virG-IncWregion (3840 bp) were amplified. The gentamycin resistance gene wascloned into the vector pCR-Blunt II-TOPO (from Invitrogen), andtransferred into E. coli TOP10 (Invitrogen) by electroporation. Thecells were incubated on an LB agar plate containing the antibioticskanamycin (50 μg/ml) at 37° C. overnight, and a plasmid was purifiedfrom the resulting colony. The nucleotide sequences of these clones(pCR-Gm) were determined by ABI PRISM Fluorescent Sequencer (Model 3100Genetic Analyzer, from Applied Biosystems) to confirm that no mutationhad been introduced by PCR error. The plasmid pCR-Gm was digested withBamHI and PvuII to recover the Gm fragment, which was ligated to thevirG-IncW fragment digested with BglII (having a BglII site at one endand a blunt end at the other). The resulting clone was transferred intoE. coli TOP10 by electroporation, and selected on an LB agar platecontaining the antibiotics gentamycin (30 μg/ml). A plasmid was purifiedfrom the resulting colony and confirmed by the sequencer to contain noPCR error, thereby generating pVGW2 (FIG. 15, SEQ ID NO: 67 in theSequence Listing).

TABLE 8 Designation Sequence Length BamSmaGmPro5′-AAA GGA TCC CGG GTT GAC ATA AGC CTG TTC GGT TCG-3′ 36 merNheIsiteGmRv 5′-AAA GCT AGC AAT TTA CCG AAC AAC TCC GCG G-3′ 31 merMscIsite-virG5′Fw 5′-AAA TGG CCA TAG GCG ATC TCC TTA ATC AAT-3′ 30 merSEQ ID NOs: 77-79 in order from the top.

Example 2 Cloning of Large Fragments by pLC Vectors

The present example describes examples of libraries of Arabidopsisthaliana (ecotype: colombia), wild species of rice (Oryza rufipogon),Sudan grass (Sorghum sudanense), an extremely early maturing variety ofItalian millet (Setaria italica), teosinte (Zea diploperennis), pearlmillet (Pennisetum typhoideum), Bahia grass (Paspalum notatum Flugge)and sugar cane (Saccharum officinarum) prepared with pLC40, pLC40GWH,pLCleo, pLC40GWHvG1, pSB200, pSB200PcHmGW, or pSB25U.

1) Preparation of Genomic DNA

About 5 g of young leaves of each plant at about one month after seedinggrown in a greenhouse was ground in a mortar under liquid nitrogen, andthen the genomic DNA was purified by the CTAB method. The yield wasabout 500-600 μg expressed as DNA. The genomic DNA was partiallydigested with 0.02-0.06 U/μg of TaqI enzyme. After the partialdigestion, fractions containing a genomic DNA fragment of 30-45 kb wererecovered by 10-40% sucrose density gradient centrifugation.

2) Preparation of the Vectors

The cosmid vectors pLC40, pLC40GWH, pLCleo, pLC40GWHvG1, pSB200,pSB200PcHmGW, and pSB25U were completely digested with the restrictionendonuclease NspV (TOYOBO) and dephosphorylated, and then purified.

3) Cloning by a Packaging Reaction

The vectors prepared as described above were ligated to the genomic DNAfragments, followed by a packaging reaction using GigaPack III XLPackaging extract at room temperature for 2 hours. After the reaction,the clones were incubated with E. coli GeneHogs (Invitrogen). As aresult, libraries of 1-100,000 cfu (colony-forming-unit) were preparedfrom all of the combinations of the plant species and vectors (Table 9),as shown in Table 7.

TABLE 9 Plant species Vector Library cfu Arabidopsis thaliana pLC40 ca80000 pSB200PcHmGWH ca 100000 Oryza rufipogon pLC40GWH ca 20000 pSB200ca 50000 Extremely early pLC40GWH ca 20000 maturing Italian milletpSB200PcHmGWH ca 20000 Sugar cane pLC40GWH ca 50000 pLC40GWHvG1 ca 50000Sudan grass pLC40GWH ca 50000 pSB200PcHmGWH ca 30000 Pearl milletpLC40GWH ca 20000 Teosinte pLC40GWH ca 100000 pSB25UNpHm ca 20000 Bahiagrass pLCleo ca 10000

4) Analysis of the Cloned Genomic DNA Fragments

Plasmids were purified from 12-24 clones of each library and cleavedwith the restriction endonucleases HindIII and SacI in the multicloningsite at each end of the insert, thereby yielding bands corresponding tothe vectors (9.2-9.8 kb) in all of the clones analyzed in the case ofpSB200, pSB25UNpHm and pSB200PcHmGW as well as bands corresponding tothe vectors (13.2-14.2 kb) in all of the clones analyzed in the case ofpLC40, pLC40GWH, pLCleo and pLC40GWHvG1. The length of the cloned largefragment is estimated to be in the range of 25 kb-45 kb from the totallength of the restriction fragments of the insert of each clone, with anaverage of about 40 kb in the case of the pSB vectors and an average ofabout 35 kb in the case of the pLC vectors. FIG. 16 shows an example ofteosinte genomic DNA/pLC40GWH.

Then, the human genome (Human Genomic DNA, Male, from Promega, CatalogNo.: G1471) was partially digested with TaqI to prepare a 30-40 kbfragment, which was then cloned into the vector pLC40GWH. Plasmid DNAwas purified from E. coli containing the human genomic fragment fromarbitrary 12 clones, and the nucleotide sequences at both ends of theinsert were analyzed and searched through a database. Homology searcheswere performed by BLAST through the database of GenBank at NCBI(http://www.ncbi.nlm.nih.gov/BLAST/). The results showed that 11 of the12 clones isolated are included in 11 single clones containing the humangenomic fragment in the database. Ten clones excluding one containingrepeated sequences were analyzed for homology to the human genomesequence in the database, whereby the lengths of the cloned humangenomic fragments were estimated to be 28023 bp, 31645 bp, 38265 bp,39599 bp, 31965 bp, 32631 bp, 34727 bp, 36925 bp, 38794 bp, and 34364bp. The average length was 34693.8 bp, which agreed well with the valueobtained by cloning the plant genomes.

Then, the nucleotide sequences at both ends of the cloned plant genomicDNA fragments were determined. Homology searches were performed by BLASTon thus obtained sequence data of 300-600 nucleotides through thedatabase of GenBank at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and thedatabase of Beijing Genomics Institute(http://btn.genomics.org.cn:8080/rice/). As a result, Oryza rufipogonand Arabidopsis showed a homology of 87-100% to the genome sequence ofrice and Arabidopsis, respectively, over the range of at least 100 by ormore. The libraries of the other plant species also showed significanthomologies to the sequences of rice, Arabidopsis, maize, sorghum, etc.

Example 3 Transfer into Agrobacterium via Triparental Mating

1) Transfer into Agrobacterium via triparental mating and its efficiency

Each vector containing a plant genomic fragment was transferred intoAgrobacterium via triparental mating as follows.

-   -   i) pLC40 series cosmid vectors

pLC40 series cosmid vectors are resistant to kanamycin (Km) andhygromycin (Hm). GeneHogs™ (Invitrogen) was used as host E. coli.pRK2073 (spectinomycin (Sp)-resistant) was used as a helper plasmid fortriparental mating. HB101 was used as host E. coli for the helperplasmid. The Agrobacterium strain LBA4404 (no drug resistance) was used.

Initially, E. coli GeneHogs™ was infected with an appropriate amount ofa dilution of a packaging reaction, spread on an LA plate containing Km(50 μg/mL), and incubated at 23° C. for 3 days. E. coli cells in acolony that appeared were streaked with a toothpick on an LA platecontaining Km, and incubated at 28° C. for 2 nights. On the other hand,LBA4404 was spread on an AB plate, and incubated at 25° C. for 5 days.HB101/pRK2073 was spread on an LA plate containing Sp (50 μg/mL), andincubated at 37° C. for 2 nights. The cultures of the three strains,i.e., GeneHogs™ harboring a pLC40 series cosmid vector containing acloned genomic fragment, LBA4404 and HB101/pRK2073 were mixed on an NAplate and incubated at 28° C. overnight. The entire amount of themixture of the three strains was suspended in 250 μl of sterile water,and 5 μl of the suspension was spread on an AB plate containing Km (50μg/mL) and Hm (25 μg/mL), and incubated at 28° C. for 7 days. Theresulting recombinant Agrobacterium was used in plant transformationexperiments. This single colony was reincubated on an AB platecontaining Km and Hm and a part of the grown colony was spread on an LAwith drugs, showing that few E. coli cells have been grown.

ii) pSB200 series cosmid vectors

pSB200 series cosmid vectors are Sp- and Hm-resistant. GeneHogs™(Invitrogen) was used as host E. coli. pRK2013 (Km-resistant) was usedas a helper plasmid. HB101 was used as host E. coli for the helperplasmid. The Agrobacterium strain LBA4404 harboring pSB1 (tetracycline(Tc) resistance) was used.

Initially, E. coli GeneHogs was infected with an appropriate amount of adilution of a packaging reaction, spread on an LA plate containing Sp(50 μg/mL), and incubated at 23° C. for 3 days. A colony was picked witha toothpick and streaked on an LA plate containing Sp, and incubated at28° C. for further 2 nights. On the other hand, LBA4404/pSB1 was spreadon an AB plate containing Tc (15 μg/mL), and incubated at 25° C. for 5days. HB101/pRK2013 was spread on an LA plate containing Km (50 μg/mL)and incubated at 37° C. for 2 nights. The cultures of the three strains,i.e., GeneHogs™ harboring a pSB200 series cosmid vector containing acloned genomic fragment, LBA4404/pSB1 and HB101/pRK2013 were mixed on anNA plate and incubated at 28° C. overnight. The entire amount of themixture of the three strains was suspended in 250 μl of sterile water,and 25 μl of the suspension was spread on an AB plate containing Sp (50μg/mL) and Hm (25 μg/mL), and incubated at 28° C. for 7 days. Theresulting recombinant Agrobacterium was used in plant transformationexperiments.

iii) pCLD04541

Two genome libraries (the genomes of the rice variety CO39 andArabidopsis ecotype Colombia, both having an average insert length of110 kb in host E. coli DH10B) prepared with the vector pCLD04541provided from Dr. Hongbin Zhang of Texas A&M University were used fortriparental mating. The pCLD04541 vector is Km- and Tc-resistant.pRK2073 was used as a helper plasmid, and HB101 was used as host E. colifor the helper plasmid. The Agrobacterium strain LBA4404 was used.

E. coli harboring each clone of the pCLD04541 libraries was spread on anLA containing Tc (10 μg/mL), and incubated at 28° C. for 2 nights. Onthe other hand, LBA4404 was spread on an AB plate, and incubated at 25°C. for 5 days. HB101/pRK2073 was spread on an LA containing Sp (50μg/mL) and incubated at 37° C. for 2 nights. The cultures of the threestrains, i.e., DH10B harboring pCLD04541 containing a cloned genomicfragment, LBA4404 and HB101/pRK2073 were mixed on an NA plate andincubated at 28° C. overnight. The entire amount of the mixture of thethree strains was suspended in 250 μl of sterile water, and a fewmicroliters of the suspension was spread on an AB plate containing Km(25 μg/mL), and incubated at 28° C. for 7 days. The resultingrecombinant Agrobacterium was used in plant transformation experiments.

As described above, genome clones included in the libraries preparedwith pLC40 series cosmid vectors, pSB200 series cosmid vectors and thepCLD04541 vector were transferred into Agrobacterium. A summary of thesetriparental mating systems and the triparental mating efficiencies areshown in Table 7. The triparental mating efficiencies were 97% in pLCseries vectors, 79% in pSB series vectors, and 93% in pCLD04541,respectively, showing that the pLC series vectors were the mostefficient (Table 10).

TABLE 10 # of clones giving # of clones recom- Effi- used for binantciency triparental Agrobact- (%) DNA donor plant Vector mating (a) erium(b) b/a <pLC40 series cosmid vectors> Oryza rufipogon pLC40 GWH 56575469 96.7 Arabidopsis thaliana pLC40 1532 1410 92.0 Sudan grass pLC40GWH 2301 2201 95.7 Italian millet pLC40 GWH 2521 2405 95.4 TeosintepLC40 GWH 10739 10593 98.6 Bahia grass pLCleo 384 383 99.7 Total 2313422461 97.1 <pSB200 series cosmid vectors > Oryza rufipogon pSB200 103757504 72.3 Arabidopsis thaliana pSB200PcHmGWH 1332 1179 88.5 Sudan grasspSB200PcHmGWH 2096 2031 96.9 Italian millet pSB200PcHmGWH 2336 2032 87.0Total 16139 12746 79.0 <pCLD04541> Indica riceCO39 pCLD04541 149 12785.2 Arabidopsis thaliana pCLD04541 192 190 99.0 Total 341 317 93.0

2) Stability of Genomic DNA

To analyze whether or not the genomic DNA fragment carried on each clonehas been transferred to Agrobacterium, Southern hybridization wasperformed using the entire genomic DNA fragment as a probe. Plasmid DNAswere conventionally extracted from E. coli and Agrobacterium, anddigested with the restriction endonucleases HindIII and SacI. Then, apart of the digests were fractionated by agarose gel electrophoresis,and transferred to the nylon membrane filter HybondN+. Then, a part ofthe HindIII and SacI digest (precipitated with ethanol and redissolvedin TE) of the E. coli-derived plasmid was labeled with an ECL labellingkit (Amersham) and hybridized to this membrane as a probe.Hybridization, washing and signal detection were performed following theinstructions attached to the ECL kit. All of four plasmids containing arufipogon fragment cloned into pLC40GWH showed the transfer of thegenomic DNA fragment from E. coli to Agrobacterium.

Example 4 Transformation of Large Fragments into Rice with pLC Vectors

1) Rice Transformation and its Efficiency

i) Method for Rice Transformation

Immature embryos of the rice variety Yukihikari were infected withAgrobacterium. Rice transformation was performed by the method describedin the Japanese Patent Application No. 2003-293125, except that all ofthe aseptically dissected immature embryos were centrifuged as apretreatment before Agrobacterium inoculation. Specifically, theimmature embryos were centrifuged in an eppendorf tube containing 1 mlof sterile water at 20000×g for 10 minutes (25° C.). Hygromycin B wasused as a selective drug and added at 50 mg/l each in the selectivemedium, regeneration medium and rooting medium. In the case of pLC40series cosmid vectors and pSB200 series cosmid vectors, one immatureembryo was inoculated with one Agrobacterium strain (one type of DNAfragment). In the case of pCLD04541, however, two immature embryos wereinoculated with one Agrobacterium strain (one type of DNA fragment).Paromomycin was used as a selective drug and added at a concentration of400-800 mg/l in the selective medium, regeneration medium and rootingmedium.

ii) Transfer of Plant Genomic Fragments into Rice

The results of transformation are shown in Table 11. In the case ofpSB200 series cosmid vectors, hygromycin-resistant individuals wereobtained from 59.1%-62.7% of the strain. In contrast, pLC40 seriescosmid vectors gave the transformants from 86.6%-95.4% of the strain. Inall of the three donor plants of genomic DNA (Oryza rufipogon, Sudangrass, and an extremely early maturing variety of Italian millet), theefficiency was 24%-36% higher when pLC40 series cosmid vectors wereused. In the case of the pCLD04541 vector, however, the efficiency wasas low as 41-53.4%. These results suggested that pLC40 series cosmidvectors allow transfer of genomic DNA fragments into rice moreefficiently than pSB200 series cosmid vectors and pLCD04541.

To evaluate the transformation efficiency of normal size gene expressionunits, vectors were tested by comparison in the transformation with aDNA fragment containing the GUS gene. When 25 Yukihikari immatureembryos were used for each vector, pSB134 (WO2005/017169) gave anaverage of 11.7 hygromycin-resistant regenerated individuals perimmature embryo while pLC40:35S-IGUS gave an average of 11.5 regeneratedindividuals.

TABLE 11 Results of the transformation of randomized plant genomicfragments into rice using a pSB or pLC vector # of # of genomic genomicfragments fragments used for that regenerated Agrobacterium- hygromycin-mediated resistant Genome donor plant Vector transformation (A)individuals (B) B/A (%) Oryza rufipogon pSB200 2246 1327 59.1 Oryzarufipogon pLC4OGWH 2271 2166 95.4 Sudan grass pSB200PcHmGWH 1997 125262.7 Sudan grass pLC4OGWH 1760 1524 86.6 Italian millet pSB200PcHmGWH1940 1200 61.9 Italian millet pLC4OGWH 2285 1986 86.9 Bahia grass pLCleo 18  16 88.9 Indica rice CO39 pCLD04541  156  64 41.0 Arabidopsisthaliana pCLD04541  189  101 53.4

2) Verification of the Transfer of Large Fragments

i) PCR of Flanking Regions of Fragments

Genomic DNAs were extracted from 11 transformants and young leaves ofYukihikari by the method described above. PCR was performed on theseDNAs with 2 sets of the primers shown in the table below. pSB200-9531Fand pSB200-4R are primers for amplifying a 139 by region from the RB tothe genomic DNA fragment. HPTinRV and HPTinFW are primers for amplifyingan internal region of the hygromycin resistance gene (Table 12).Thirty-five cycles of PCR were performed. As a result, the products wereamplified with HPTinRV and HPTinFW in all of the 11 transformants, whileno PCR product was obtained with either primer set in the controlYukihikari. When pSB200-9531F and pSB200-4R were used, PCR products wereobtained in 10 of the 11 individuals. These results show that theflanking regions of the genomic DNA fragments were transferred into mostof the plants transformed with pLC vectors, thus verifying the transferof the genomic DNA fragments.

TABLE 12 Designation Sequence Length pSB200-9531F5′-ctg aag gcg gga aac gac aat ctg-3′ 24 mer pSB200-4R5′-gct tgc tga gtg gct cct tca acg-3′ 24 mer pSB200-170R5′-aac tgc act tca aac aag tgt gac-3′ 24 mer HPTinRV5′-tat gtc ctg cgg gta aat ag-3′ 20 mer HPTinFW5′-ttg ttg gag ccg aaa tcc g-3′ 19 mer SEQ ID NOs: 60-64 in order fromthe top.

ii) PCR of Both Terminal and Internal Sequences of Fragments

For each of three Oryza rufipogon fragments (called A, B, and C) usedfor the transformation into Yukihikari with the pLC40GWH vector, twoindividuals of TO plant were analyzed by PCR to determine whether or notboth ends and the center region of each fragment had been introduced.PCR conditions included a treatment at 94° C. for 2 minutes, followed by35 cycles of thermal denaturation at 94° C. for 30 seconds, annealing at60° C. for 30 seconds and extension at 60° C. for 30 seconds, andfinally a treatment at 72° C. for 2 minutes.

To detect the RB side of fragment A, PCR (PCR1) was performed withpSB200-9531F and a primer specific to fragment A (5′-gtt aat ttc ttg tgatcg aag gac-3′ (SEQ ID NO: 11)). To detect the center region of fragmentA, a PCR assay was performed by the CAPS method (Konieczny and Ausubel1993 Plant Journal 4: 403-410) using nucleotide sequence polymorphismsfound between the sequence of Nipponbare AP004667 corresponding tofragment A (identified by database searches) and the sequence of Oryzarufipogon. Specifically, PCR (PCR2) was performed with two primers(5′-ggg att ctt tat gct ggg ttt agg-3′ (SEQ ID NO: 12) and 5′-gca agcaat acc tct gtt atg ctg-3′ (SEQ ID NO: 13)), and the product wasdigested with SspI. To detect the HPT side, PCR (PCR3) was performedwith pSB200-170R and a primer specific to fragment A (5′-gtt ttc aga tggcga cct cag ctt tg-3′ (SEQ ID NO: 14)).

Similar marker assays were performed on fragment B and fragment C. Thus,to detect the RB side of fragment B, PCR was performed with pSB200-9531Fand a primer specific to fragment B (5′-cag gtg gct tta ttc ctc ctctca-3′ (SEQ ID NO: 15)). To detect the center region of fragment B, aPCR assay was performed by the CAPS method using nucleotide sequencepolymorphisms found between the sequence of Nipponbare AP005967corresponding to fragment B (identified by database searches) and thesequence of Oryza rufipogon. Specifically, PCR was performed with twoprimers (5′-ccg aaa gtt cgt ggg caa tgc cta-3′ (SEQ ID NO: 16) and5′-gcc atc ctt agc ata tga gtg gca-3′ (SEQ ID NO: 17)), and the productwas digested with HaeIII. To detect the HPT side of fragment B, PCR wasperformed with pSB200-170R and a primer specific to fragment B (5′-ggctat tta cgt ggc atg tta cgt-3′ (SEQ ID NO: 18)). To detect the RB sideof fragment C, PCR was performed with pSB200-9531F and a primer specificto fragment C (5′-tcg taa gtc tac ttc cct tta cga-3′ (SEQ ID NO: 19)).To detect the center region of fragment C, a PCR assay was performed bythe CAPS method using nucleotide sequence polymorphisms found betweenthe sequence of Nipponbare AL713907 corresponding to fragment C(identified by database searches) and the sequence of Oryza rufipogon.Specifically, PCR was performed with two primers (5′-cca aac cac atc cttata gtg tgc-3′ (SEQ ID NO: 20) and 5′-cct cat tgc atg cgg tca cta c-3′(SEQ ID NO: 21)), and the product was digested with HaeIII. To detectthe HPT side of fragment C, PCR was performed with pSB200-170R and aprimer specific to fragment C (5′-gca ggg tat taa tcg atc aac acc-3′(SEQ ID NO: 22)).

Analytical results of fragment B are shown in FIG. 17, and analyticalresults of fragments A-C are summarized in Table 13. Of the twotransformants tested for fragment A, no individual containing the entirelarge fragment was obtained but an individual containing the centerregion and one end, or both ends was obtained. However, one of the twoindividuals tested for fragment B and fragment C was shown to containthe entire Oryza rufipogon fragment, i.e., both ends and the centerregion. These results verified that plant genomic fragments of 25-40 kbin size can be transferred into plants by pLC vectors.

TABLE 13 Fragment T0 plant RB side center HPT side A 1 − + + 2 + − + B1 + + − 2 + + + C 1 − − − 2 + + + +: An oryza rufipogon fragment wasdetected. −: An oryza rufipogon fragment was not detected.

Example 5 Transformation of Maize with pLC40 Series Cosmid Vectors

1) Combination of pLC with pTOK47 or pLC with pVGW

A vector containing a vir gene is required for maize transformation toincrease the transformation efficiency (Ishida et al. 1996 NatBiotechnol 14:745-50) because the efficiency with ordinary binaryvectors is very low except for special methods (Frame et al. (2002)Plant Physiol 129: 13-22). pLC40 series cosmid vectors are ordinarybinary vectors so that they should be modified by using a vir gene toimprove the transformation efficiency. Thus, the vector pTOK47 capableof coexisting with pLC40 series cosmid vectors (IncP plasmids) inbacteria and expressing a vir gene (Jin et al. 1987 J Bacteriol 169:4417-4425), and a vector newly constructed by the present invention,pVGW were initially used in combination with pLC. pTOK47 is an IncWplasmid carrying a DNA fragment (KpnI 14.8 kb fragment) containing thevirB gene and the virG gene derived from the Agrobacterium strain A281,and capable of coexisting with IncP plasmids. pVGW is a plasmidcontaining a variant virG (virGN54D) and IncW ori.

pTOK47 (tetracycline-resistant) was transferred into the AgrobacteriumLBA4404 or EHA105 (a kind gift from Dr. Stanton Gelvin of PurdueUniversity) via triparental mating. A plasmid was extracted from thisAgrobacterium and confirmed by restriction endonuclease analysis tocontain pTOK47. Further, pLC40:35S-IGUS or pLC40GWB:35S-IGUS wasintroduced into the resulting LBA4404/pTOK47 or EHA105/pTOK47(Tc-resistant) via triparental mating. These Agrobacteria are describedas LBA4404/pTOK47/pLC40:35S-IGUS, LBA4404/pTOK47/pLC40GWB:35S-IGUS,EHA105/pTOK47/pLC40:35S-IGUS, and EHA105/pTOK47/pLC40GWB:35S-IGUS.Plasmid DNAs were extracted from the Agrobacteria and analyzed by PCR toconfirm the presence of the VirG, RB, hpt or bar, and GUS genes.

In the same manner, pVGW was transferred into the Agrobacterium LBA4404by electroporation, and a colony was selected by gentamycin (Gm 50μg/mL). pLC40:35S-IGUS or pLC40GWB:35S-IGUS was introduced into theresulting LBA4404/pVGW via triparental mating. These Agrobacteria aredescribed as LBA4404/pVGW/pLC40:35S-IGUS andLBA4404/pVGW/pLC40GWB:35S-IGUS. Agrobacterium colonies (Km- andGm-resistant) were directly analyzed by PCR to confirm the presence ofthe VirG, hpt or bar, and GUS genes.

Moreover, pIG121Hm derived from the IncP plasmid pBI121 (Hiei et al.(1994) Plant J 6: 271-282) was introduced into LB4404/pTOK47 to prepareAgrobacterium LB4404/pTOK47/pIG121Hm, which was used as a control inmaize transformation experiments.

2) Transformation of Maize

Maize immature embryos having a size of about 1.2 mm (variety: A188)were aseptically removed from a plant grown in a greenhouse, andimmersed in a liquid medium for suspending Agrobacterium (LS-inf, Ishidaet al. 1996). After thermal treatment at 46° C. for 3 minutes, theimmature embryos were washed with the same liquid medium. Aftercentrifugation at 15,000 rpm, 4° C., for 10 minutes, the immatureembryos were then immersed in a suspension of each strain at about 1×10⁹cfu/ml in LS-inf medium (containing 100 μM acetosyringon) and thenplated on a coculture medium (LS-AS (Ishida et al. 1996 Nat Biotechnol14:745-50) containing AgNO₃, CuSO₄). After incubation at 25° C. indarkness for 3 days, the immature embryos were partially used for GUSanalysis.

The cocultured immature embryos were plated on a selective mediumcontaining hygromycin or phosphinothricin (Ishida et al. (2003) PlantBiotechnology 20:57-66) and incubated. A callus grown was excised andplated on a regeneration medium containing hygromycin (Hm) orphosphinothricin (PPT) (Ishida et al. 1996 Nat Biotechnol 14:745-50),and incubated under illumination. After two weeks, regenerated plantsshowing resistance to Hm or PPT were investigated.

Initially, A188 immature embryos were inoculated with various strainsand observed for the transient expression of the GUS gene on day 3 ofcoculture. Immature embryos inoculated with the control LBA4404/pSB134showed the expression of the GUS gene over a wide range. However, fewimmature embryos inoculated with LBA4404/pLC40:35S-IGUS showed theexpression except for limited ones showing the expression in very smallspots. No increase in expression was found when EHA105 was used as ahost. Most of immature embryos inoculated withLBA4404/pTOK47/pLC40:35S-IGUS, LBA4404/pLC40GWHvG1:35S-IGUS,LBA4404/pVGW/pLC40:35S-IGUS and LBA4404/pVGW/pLC40GWB:35S-IGUS showedspots representing the expression of the GUS gene to a lesser extentthan with LBA4404/pSB134, thus verifying that the gene transferefficiency is improved by the coexistence with a plasmid containing thevirB gene and virG gene derived from the Agrobacterium strain A281, orthe coexistence with a plasmid containing virGN54D, or the addition ofthe virG gene. On the other hand, there is no difference in theexpression of the GUS gene between pLC40GWHvG1:35S-IGUS andpLC40GWHvGC1:35S-IGUS, showing that a single nucleotide substitution forremoving an NspV recognition site does not influence the virG activity.

Then, we tried to create transformed plants by incubating the coculturedimmature embryos in a selective medium containing Hm or PPT and aregeneration medium. When EHA105 was used as a host, the pLCSBGWBSWvector gave no PPT-resistant plant. When LBA4404 was used as a host,however, the pLCSBGWBSW vector gave plants showing resistance to PPT atan efficiency comparable to that of the superbinary vector pSB131(containing the GUS gene and the bar gene in the T-DNA region, Ishida etal. 1996 Nat Biotechnol 14:745-50) using the same strain as a host(Table 14).

LBA4407/pTOK47/pLC40GWB:35S-IGUS was also shown to give PPT-resistantplants at a high efficiency comparable to that of the superbinary vectorpSB131. When the hygromycin resistance gene was used as a selectablemarker gene, a pLC40 series cosmid vector (pLC40:35S-IGUS) combined withpTOK47 also gave hygromycin-resistant plants (Table 13). pLC40GWHvG1containing the virG gene also achieved an efficiency comparable to thatof the superbinary vector SB134 (containing the GUS gene and thehygromycin resistance gene in the T-DNA region, Hiei and Komari 2006Plant Cell, Tissue and Organ Culture 85: 271-283) (Table 14).

TABLE 14 Results of transformation of maize Rediffer- # of immatureembryos entiation Exper- Selective Inoculated rediffer- ratio imentStrain drug (A) entiated (B) (B/A, %) 1 LBA4404 (pLCSBGWBSW) PPT 46 1021.7 EHA105 (pLCSBGWBSW) PPT 46  0 0 LBA4404 (pSB131) PPT 45  9 20.0 2LBA4404 (pLC40GWB:35S-IGUS) PPT 56  0 0 LBA4404 (pLC40GWB:35S-IGUS/ PPT57 14 24.6 pTOK47) LBA4404 (pSB131) PPT 59 19 32.2 3LBA4404(pLC40:35S-IGUS) Hm 43  0 0 LBA4404(pLC40:35S-IGUS /pTOK47) Hm 44 2 4.5 LBA4404(pIG121Hm) Hm 42  0 0 LBA4404(pIG121Hm/pTOK47) Hm 42  0 04 LBA4404(pLC40GWHvG1) Hm 59  5 8.5 LBA4404(pSB134) Hm 57  5 8.8 PPT:phosphinothricin, Hm: hygromicin

In order to examine the influence of pVGW on maize transformation, maizewas then transformed with LBA4404/pLC40:35S-IGUS,LBA4404/pVGW/pLC40:35S-IGUS, LBA4404/pVGW/pLC40GWB:35S-IGUS, andLBA4404/pSB134, and the regenerated individuals were analyzed for GUSexpression. As a result, the proportion of the number of GUS-expressingindividuals in pLC40:35S-IGUS was 0% (0/16), while the proportion of thenumber of GUS-expressing individuals per inoculated immature embryo inpLC40:35S-IGUS and pLC40GWB:35S-IGUS both combined with pVGW reached 40%(6/15) and 30% (6/20), respectively, which were comparable to 41.2%(7/17) in the superbinary vector pSB134. Thus, the transformation ofmaize with pLC vectors could be achieved at high efficiency by usingpVGW.

We further tried to transform plant genomic fragments into maize bycombining a pLC vector and the pVGW vector. A genomic fragment (30-35kb) of Sudan grass was randomly cloned into the NspV site of the vectorpLC40GWB. The resulting E. coli plasmid was transferred to Agrobacteriumharboring pVGW (LBA4404) via triparental mating. In this manner,Agrobacterium harboring both of the plasmids pLC40GWB containing thegenomic fragment of Sudan grass and pVGW was prepared, and inoculatedinto maize immature embryos (variety: A188). Transformed cells wereselected to show that 17 of the 27 fragments inoculated gaveredifferentiated plants (Table 15). This showed that plant genomicfragments can be efficiently transformed into maize by the combinationof pLC and pVGW.

These results demonstrated that maize transformation can be efficientlyachieved by the combination with a plasmid carrying a DNA fragmentcontaining the virB gene and virG gene derived from the Agrobacteriumstrain A281 such as pTOK47, or the combination with a plasmid containingthe virGN54D gene such as pVGW, or the incorporation of the virG geneinto a pLC vector such as pLC40GWHvG1.

TABLE 15 Results of the transformation of randomized plant genomicfragments into maize using a pLC/pVGW vector system # of genomic # ofgenomic fragments used for fragments that Agrobacterium- regeneratedGenome mediated PPT-resistant B/A donor plant Strain transformation (A)individuals (B) (%) Sudan grass LBA4404(pLC40GWB/pVGW) 27 17 63.0

Example 6 Isolation of a Gene of Interest from BAC Clones Using pLCVectors

Komori et al. (2004) (Plant J 37: 315-325) found that a cytoplasmic malesterile strain restores fertility when it is transformed with the PPR791gene isolated from the rice variety IR24, thus demonstrating that PPR791is the fertility restorer gene Rf-1. The PRR791 gene was identical withthe PPR8-1 gene of the rice variety Milyang 23 that had been previouslyreported as a candidate for Rf-1 by Kazama and Toriyama (2003) (FEESLett 544: 99-102). Thus, the BAC clone OSIMBb0046F08 of Milyang 23 fromwhich the PPR8-1 gene had been derived was obtained from ClemsonUniversity, and a model experiment was performed for isolating Rf-1 fromthe BAC.

Initially, a plasmid was extracted from OSIMBb0046F08 using High PurityPlasmid Midiprep System (Marligen). The plasmid was partially digestedwith TaqI and a DNA fragment around 30 kb was recovered by sucrosedensity gradient centrifugation. This DNA fragment was ligated to theBstBI-digested and CIP-treated pLC40GWH vector or the BstBI-digested andCIP-treated pSB200 vector using DNA Ligation Kit <Mighty Mix> (TakaraBio Inc.). The resulting construct was transferred into E. coli byelectroporation to give colonies of transformants on an LB platecontaining an appropriate antibiotic (50 μg/ml kanamycin orspectinomycin). To determine the presence or absence of the Rf-1 gene inthe resulting plasmid, direct PCR (see Examples 1, 3)) was performed byusing these colonies as templates along with primers designed for theRf-1 gene (WSF7T7R1 and IR50226R, Table 16) to select Rf-1 positiveclones giving an amplified product of about 2 kb from Rf-1 negativeclones showing no amplification of the product. The incidence ofpositive clones in this PCR screening was 5/39 (12.8%) in the pLC40GWHconstruct and 6/96 (6.3%) in the pSB200 construct. That is, the cloningefficiency of a gene of interest was about twice higher in the pLCvector than pSB.

One positive clone and two negative clones selected from the pLC40GWHconstruct, and one positive clone and two negative clones selected fromthe pSB200 construct were transferred from E. coli to Agrobacterium viatriparental mating. The cytoplasmic male sterile strain MS Koshihikariwas infected with the resulting Agrobacterium by the method described inKomori et al. (2004). The resulting transformed rice was acclimated andthen grown in a greenhouse. During the maturing stage, an average earwas collected from each individual and evaluated for the fertility rate.The results showed that transformants from constructs containing no Rf-1(pLC-7, pLC-11, pSB-1, pSB-7) were sterile, while constructs containingRf-1 (pLC-8, pSB-37) gave fertile transformants (Table 17).

These results demonstrated that a gene of interest can be efficientlyidentified by preparing a library from DNA of BAC containing the gene ofinterest using a cosmid vector for plant transformation and transferringit into a plant and then selecting a plant showing an expectedphenotype.

TABLE 16 Primer Name Sequence Length WSF7T7R15′-AGT GTG TGG CAT GGT GCA TTT 24 mer CCG-3′ IR50226R5′-CTC TAC AGG ATA CAC GGT GTA 24 mer AGG-3′ SEQ ID NOs: 80-81 in orderfrom the top.

TABLE 17 Fertility restoration by various constructs Presence(+) or # of# of absence(−) individuals individuals Construct of Rf-1 analyzedfetile pLC-8 + 6 4 pLC-7 − 9 0 pLC-11 − 8 0 pSB-37 + 9 6 pSB-1 − 9 0pSB-7 − 9 0

In conclusion, pLC vectors are characterized in that:

1. they allow easy cloning of DNA in the order of 25-40 kb;2. they are stable in bacteria; and3. they allow efficient transformation of plants, especiallymonocotyledons.

pLC vector series are useful for handling medium-size DNA in the fieldof functional genomics.

1. A cosmid vector having a full length of 15 kb or less characterizedin that: 1) it contains an origin of replication (oriV) of an IncPplasmid, but does not contain any origin of replication of other plasmidgroups; 2) it contains the trfA1 gene of an IncP plasmid; 3) it containsan origin of conjugative transfer (oriT) of an IncP plasmid; 4) itcontains the incC1 gene of an IncP plasmid; 5) it contains a cos site oflambda phage and the cos site is located outside the T-DNA; 6) itcontains a drug resistance gene expressed in E. coli and a bacterium ofthe genus Agrobacterium; 7) it contains a T-DNA right border sequence ofa bacterium of the genus Agrobacterium; 8) it contains a T-DNA leftborder sequence of a bacterium of the genus Agrobacterium; 9) itcontains a selectable marker gene for plant transformation locatedbetween 7) and 8) and expressed in a plant; and 10) it containsrestriction endonuclease recognition site(s) located between 7) and 8)for cloning a foreign gene, wherein the cosmid vector is selected fromthe group consisting of: the cosmid vector pLC40 consisting of thenucleotide sequence of SEQ ID NO: 2 or an equivalent thereof consistingof a nucleotide sequence having at least 95% identity to the nucleotidesequence; the cosmid vector pLC40GWH consisting of the nucleotidesequence of SEQ ID NO: 3 or an equivalent thereof consisting of anucleotide sequence having at least 95% identity to the nucleotidesequence; the cosmid vector pLC40 bar consisting of the nucleotidesequence of SEQ ID NO: 4 or an equivalent thereof consisting of anucleotide sequence having at least 95% identity to the nucleotidesequence; the cosmid vector pLC40GWB consisting of the nucleotidesequence of SEQ ID NO: 5 or an equivalent thereof consisting of anucleotide sequence having at least 95% identity to the nucleotidesequence; the cosmid vector pLC40GWHKorB consisting of the nucleotidesequence of SEQ ID NO: 65 or an equivalent thereof consisting of anucleotide sequence having at least 95% identity to the nucleotidesequence; the cosmid vector pLCleo consisting of the nucleotide sequenceof SEQ ID NO: 66 or an equivalent thereof consisting of a nucleotidesequence having at least 95% identity to the nucleotide sequence; andthe cosmid vector pLC40GWHvG1 consisting of the nucleotide sequence ofSEQ ID NO: 7 or an equivalent thereof consisting of a nucleotidesequence having at least 95% identity to the nucleotide sequence;wherein the cosmid vector is stably maintained in E. coli andAgrobacterium cells.
 2. A method for transforming a plant, comprisingtransforming the plant with a bacterium of the genus Agrobacteriumharboring an expression vector containing a nucleic acid fragment of aplant inserted into the cosmid vector of claim
 1. 3. A method fortransforming a plant, comprising transforming the plant with a bacteriumof the genus Agrobacterium harboring the cosmid vector according toclaim 1 and a plasmid vector characterized in that: 1) it contains anelement necessary for the replication of an IncW plasmid, but does notcontain any origin of replication of other plasmid groups; 2) itcontains the repA gene necessary for the replication of an IncW plasmid;3) it contains a drug resistance gene expressed in E. coli and abacterium of the genus Agrobacterium; and 4) the virG gene of abacterium of the genus Agrobacterium.
 4. The method for transforming aplant according to claim 2 or 3, wherein the selectable marker gene forplant transformation is selected from the group consisting of ahygromycin resistance gene, a phosphinotricin resistance gene and akanamycin resistance gene.